Bispecific CD33 and CD3 Binding Proteins

ABSTRACT

Described herein are binding proteins that specifically bind to human CD33, and in particular to bispecific binding proteins that specifically bind to human CD33 and human CD3. Also described herein are bispecific tandem diabodies that bind to CD33 and CD33, and their uses for immunotherapy of CD33+ cancers, diseases and conditions such as acute myeloid leukemia (AML).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/937,494, filed Nov. 10, 2015, which is a continuation of U.S. patentapplication Ser. No. 14/642,497, filed Mar. 9, 2015 (now U.S. Pat. No.9,212,225, issued Jan. 7, 2016), and claims the benefit of U.S.Provisional Application No. 62/111,470, filed Feb. 3, 2015, and U.S.Provisional Application No. 62/019,795, filed Jul. 1, 2014, all of whichare incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Acute myeloid leukemia (AML) is an acute leukemia in adults andchildren. CD33 is expressed on the majority of myeloblasts in AML. CD33,in some reports, is generally restricted to early multilineage myeloidprogenitors and absent from normal pluripotent hematopoietic stem cells.

SUMMARY OF THE INVENTION

Provided herein are binding proteins that specifically bind to humanCD33, and bispecific binding proteins that specifically bind to humanCD33 and human CD3. Also provided herein are anti-CD33 variable domainsand anti-CD3 variable domains for generating a number of bispecificCD33/CD3 binding proteins, such as, for example, tandem diabodies. Alsofurther provided herein are bispecific tandem diabodies that bind toCD33 and CD3 and their use for immunotherapy of acute myeloid leukemia(AML) and other hematologic malignancies, disorders or conditions.

In particular, the binding proteins are provided that show binding toboth human as well as cynomolgus monkey CD33. It is demonstrated in theexamples that these CD33/CD3 tandem diabodies can re-direct polyclonalCD3⁺ T-cells from healthy donors, as well as autologous T-cells from AMLpatients, to effectively lyse CD33⁺ AML cells at low E:T cell ratios. Inthis process, which is dependent on the presence of both CD33⁺ targetcells and T-cells, re-directed T-cells are activated, as shown byinduction of CD25 and CD69, and stimulated to proliferate. The anti-AMLeffect of these tandem diabodies is shown to be dependent on theconcentration of the antibodies used as well as on the E:T cell ratio.The tandem diabody is tetravalent and has two binding sites for CD33 andtwo binding sites for CD3. A particular feature of the CD33/CD3 tandemdiabodies described herein is that they facilitate potent and efficientapoptosis as a result of bivalent binding that confers avidity to eachantigen, namely CD33 and CD3.

In summary, the provided CD33/CD3 binding proteins described herein, inparticular tandem diabodies, induce potent cytolysis of CD33⁺ leukemiccells and primary AML cells in vitro. Examples of bispecific CD33/CD3binding proteins in the antibody format of tandem diabodies demonstratecytolytic activity in vivo in cell lines, primary AML cells and in invivo models with AML cell lines and with patient derived primary AMLcells. This indicates high in vivo activity especially noteworthy in thestringent AML PDX model. Further, examples of bispecific CD33/CD3binding proteins in the antibody format of tandem diabodies demonstratecytolytic activity ex vivo in samples from patients at all stages ofAML, including newly diagnosed, relapsed and refractory patients.

Furthermore, these CD33/CD3 binding proteins described herein are ableto achieve a significant lysis of CD33 expressing cells within aboutfour hours. CD33/CD3 binding proteins accordingly exhibit highcytotoxicity at low CD33 densities on the cell surface as well as a highcytotoxicity at low effector: target (E:T) ratios. In addition, CD33/CD3binding proteins described herein exhibit not only potent CD33 and CD3binding affinities to the human proteins, but show also excellentcrossreactivity with the respective cynomolgus monkey proteins, forexample with human:cynomolgous K_(D) ratios between 5 and 0.2.Furthermore, the CD33/CD3 binding proteins described herein show nosignificant induction of cytokine release in the absence of CD33⁺ targetcells which is an essential component of the safety profile of thesemolecules. Moreover, the CD33/CD3 tandem diabodies described hereinbelong to the class of molecules that have half-lives in the approximaterange of 8-24 h, which should allow convenient dosing.

In one aspect, provided herein are CD33 binding proteins thatspecifically bind to an epitope of human CD33. In some embodiments, thebinding proteins comprise a heavy chain variable domain and a lightchain variable domain that is derived from human.

In some embodiments, a CD33 binding protein has at least one bindingsite comprising a light chain variable domain and a heavy chain variabledomain, wherein the light chain variable domain comprises a CDR1consisting of the sequence selected from the group consisting of SEQ IDNOs:21-27, a CDR2 consisting of the sequence selected from the groupconsisting of SEQ ID NOs:28-34 and a CDR3 consisting of the sequence ofthe group consisting of SEQ ID NOs:35-41.

In some embodiments, a CD33 binding protein has at least one bindingsite comprising a light chain variable domain and a heavy chain variabledomain, wherein the heavy chain variable domain comprises a CDR1consisting of the sequence selected from the group consisting of SEQ IDNOs:42-48, a CDR2 consisting of the sequence selected from the groupconsisting of SEQ ID NOs:49-55 and a CDR3 consisting of a sequencesselected from the group consisting of SEQ ID NOs:56-63.

In certain instances, the CDR1, CDR2 and CDR3 of the light chainvariable domain is selected from the group consisting of SEQ ID NOs:21,28 and 35; SEQ ID NOs:22, 29 and 36; SEQ ID NOs:23, 30 and 37; SEQ IDNOs:24, 31 and 38; SEQ ID NOs:25, 32 and 39; SEQ ID NOs:26, 33 and 40;and SEQ ID NOs:27, 34 and 41.

In certain instances, the CDR1, CDR2 and CD3 of the heavy chain variabledomain is selected from the group consisting of SEQ ID NOs:42, 49 and56; SEQ ID NOs:43, 50 and 57; SEQ ID NOs:43, 50 and 58; SEQ ID NOs:43,50 and 59; SEQ ID NOs:43, 50 and 60; SEQ ID NOs:44, 51 and 61; SEQ IDNOs:45, 52 and 62; SEQ ID NOs:46, 53 and 63; SEQ ID NOs:47, 54 and 63;and SEQ ID NOs:48, 55 and 63.

In certain instances, the human CD33 binding site of a variable heavychain domain and a variable light chain domain is selected from thegroup consisting of SEQ ID NO: 1 and SEQ ID NO: 11; SEQ ID NO:2 and SEQID NO: 12; SEQ ID NO:3 and SEQ ID NO: 13; SEQ ID NO:4 and SEQ ID NO:14;SEQ ID NO:5 and SEQ ID NO:15; SEQ ID NO:6 and SEQ ID NO:16; SEQ ID NO:7and SEQ ID NO:17; SEQ ID NO:8 and SEQ ID NO:18; SEQ ID NO:9 and SEQ IDNO:19; and SEQ ID NO: 10 and SEQ ID NO:20.

In some embodiments, the CD33 epitope is within ₆₂DQEVQEETQ₇₀ (SEQ IDNO:94) amino acid residues 62-70 of SEQ ID NO:93) of human CD33.

In any of the above embodiments, the CD33 binding protein comprises atleast one further functional domain. In some instances, the functionaldomain is an effector domain that binds to an effector cell. In certaininstances, the effector domain is a CD3 binding site comprising at leastone antibody variable heavy chain domain and at least one variable lightchain domain forming an antigen binding site for human CD3.

In certain instances, the CD3 binding site comprises a heavy chainvariable domain comprising a CDR1 sequence of STYAMN (SEQ ID NO:72), aCDR2 sequence of RIRSKYNNYATYYADSVKD (SEQ ID NO:73) and a CDR3 sequenceof HGNFGNSYVSWFAY (SEQ ID NO:74). In other instances, the CD3 bindingsite comprises a light chain variable domain comprising a CDR1 sequenceofRSSTGAVTTSNYAN (SEQ ID NO:90), a CDR2 sequence of GTNKRAP (SEQ IDNO:91), and a CDR3 sequence of ALWYSNL (SEQ ID NO:92).

In certain instances, the CD3 binding site comprises a heavy chainvariable domain of SEQ ID NO:64 and a variable light chain domain of SEQID NO:68; a heavy chain variable domain of SEQ ID NO:65 and a variablelight chain domain of SEQ ID NO:69; a heavy chain variable domain of SEQID NO:66 and a variable light chain domain of SEQ ID NO:70; or a heavychain variable domain of SEQ ID NO:67 and a variable light chain domainof SEQ ID NO:71.

In any of the above embodiments, the CD33 binding protein is a dimericprotein. In any of the above embodiments, the CD33 binding protein ismultifunctional.

In certain instances, the multifunctional CD33 binding protein hasbispecificity for CD33 and CD3, wherein the binding specificities areprovided by heavy chain variable domain and light chain variable domainsfor CD33 and CD3 selected from the group consisting of SEQ ID NOs:2, 12,65 and 69; SEQ ID NOs:3, 13, 65 and 69; SEQ ID NOs:4, 14, 65 and 69; SEQID NOs:5, 15, 65 and 69; SEQ ID NOs: 1, 11, 64 and 68; SEQ ID NOs:2, 12,64 and 68; SEQ ID NOs:2, 12, 66 and 70; SEQ ID NOs:4, 14, 66 and 70; SEQID NOs:5, 15, 66 and 70; SEQ ID NOs:3, 13, 64 and 68; SEQ ID NOs:3, 13,67 and 71; SEQ ID NOs:4, 14, 64 and 68; SEQ ID NOs:5, 15, 64 and 68; SEQID NOs:7, 17, 64 and 68; SEQ ID NOs:6, 16, 64 and 68; SEQ ID NOs:6, 16,67 and 71; SEQ ID NOs:8, 18, 64 and 68; SEQ ID NOs:9, 19, 64 and 68; SEQID NOs:9, 19, 67 and 71; and SEQ ID NOs:10, 20, 64 and 68.

In another aspect, provided herein are bispecific, antigen-bindingtandem diabodies specific to human CD3 and human CD33. In someembodiments, the tandem diabodies comprise a first polypeptide and asecond polypeptide, each polypeptide having at least four variable chaindomains linked one after another, wherein each polypeptide comprises avariable heavy chain domain specific for human CD33; a variable lightchain domain specific for human CD33; a variable heavy chain domainspecific for human CD3, and a variable light chain domain specific forhuman CD3 and wherein in each polypeptide the four variable chaindomains are linked with one after another by peptide linkers L1, L2 andL3 in the order of VL(CD3)-L1-VH(CD33)-L2-VL(CD33)-L3-VH(CD3);VH(CD3)-L1-VL(CD33)-L2-VH(CD33)-L3-VL(CD3);VL(CD33)-L1-VH(CD3)-L2-VL(CD3)-L3-VH(CD33); orVH(CD33)-L1-VL(CD3)-L2-VH(CD3)-L3-VL(CD33).

In some embodiments, the VL domain specific to human CD33 comprises aCDR1 consisting of the sequence selected from the group consisting ofSEQ ID NOs:21-27, a CDR2 consisting of the sequence selected from thegroup consisting of SEQ ID NOs:28-34 and a CDR3 consisting of thesequence of the group consisting of SEQ ID NOs:35-41.

In some embodiments, the VH domain specific to human CD33 comprises aCDR1 consisting of the sequence selected from the group consisting ofSEQ ID NOs:42-48, a CDR2 consisting of the sequence selected from thegroup consisting of SEQ ID NOs:49-55 and a CDR3 consisting of asequences selected from the group consisting of SEQ ID NOs:56-63.

In some embodiments, the CDR1, CDR2 and CDR3 of the VL domain specificto human CD33 are sequences selected from the group consisting of SEQ IDNOs:21, 28 and 35; SEQ ID NOs:22, 29 and 36; SEQ ID NOs:23, 30 and 37;SEQ ID NOs:24, 31 and 38; SEQ ID NOs:25, 32 and 39; SEQ ID NOs:26, 33and 40; and SEQ ID NOs:27, 34 and 41.

In some embodiments, the CDR1, CDR2 and CDR3 of the VH domain specificto human CD33 are sequences selected from the group consisting of SEQ IDNOs:42, 49 and 56; SEQ ID NOs:43, 50 and 57; SEQ ID NOs:43, 50 and 58;SEQ ID NOs:43, 50 and 59; SEQ ID NOs:43, 50 and 60; SEQ ID NOs:44, 51and 61; SEQ ID NOs:45, 52 and 62; SEQ ID NOs:46, 53 and 63; SEQ IDNOs:47, 54 and 63; and SEQ ID NOs:48, 55 and 63.

In some embodiments, the VL and VH domains specific to CD33 aresequences selected from the group consisting of SEQ ID NO: 1 and SEQ IDNO: 11; SEQ ID NO:2 and SEQ ID NO: 12; SEQ ID NO:3 and SEQ ID NO: 13;SEQ ID NO:4 and SEQ ID NO: 14; SEQ ID NO:5 and SEQ ID NO:15; SEQ ID NO:6and SEQ ID NO:16; SEQ ID NO:7 and SEQ ID NO:17; SEQ ID NO:8 and SEQ IDNO:18; SEQ ID NO:9 and SEQ ID NO:19; and SEQ ID NO:10 and SEQ ID NO:20.

In some embodiments, the VH domain specific for human CD3 comprises aCDR1 sequence of STYAMN (SEQ ID NO:72), a CDR2 sequence ofRIRSKYNNYATYYADSVKD (SEQ ID NO:73) and a CDR3 sequence of HGNFGNSYVSWFAY(SEQ ID NO:74) or HGNFGNSYVSYFAY (SEQ ID NO:75).

In some embodiments, the VL domain specific for human CD3 comprises aCDR1 sequence of RSSTGAVTTSNYAN (SEQ ID NO:90), a CDR2 sequence ofGTNKRAP (SEQ ID NO:91), and a CDR3 sequence of ALWYSNL (SEQ ID NO:92).

In some embodiments, the VL and VH domains specific to CD3 are sequencesselected from the group consisting of SEQ ID NO:64 and SEQ ID NO:68; SEQID NO:65 and SEQ ID NO:69; SEQ ID NO:66 and SEQ ID NO:70; and SEQ IDNO:67 and SEQ ID NO:71.

In some embodiments, each polypeptide comprises four variable chaindomains selected from the group consisting of SEQ ID NOs:2, 12, 65 and69; SEQ ID NOs:3, 13, 65 and 69; SEQ ID NOs:4, 14, 65 and 69; SEQ IDNOs:5, 15, 65 and 69; SEQ ID NOs: 1, 11, 64 and 68; SEQ ID NOs:2, 12, 64and 68; SEQ ID NOs:2, 12, 66 and 70; SEQ ID NOs:4, 14, 66 and 70; SEQ IDNOs:5, 15, 66 and 70; SEQ ID NOs:3, 13, 64 and 68; SEQ ID NOs:3, 13, 67and 71; SEQ ID NOs:4, 14, 64 and 68; SEQ ID NOs:5, 15, 64 and 68; SEQ IDNOs:7, 17, 64 and 68; SEQ ID NOs:6, 16, 64 and 68; SEQ ID NOs:6, 16, 67and 71; SEQ ID NOs:8, 18, 64 and 68; SEQ ID NOs:9, 19, 64 and 68; SEQ IDNOs:9, 19, 67 and 71; and SEQ ID NOs:10, 20, 64 and 68.

In some embodiments, linkers L1, L2 and L3 consist of about 12 or lessamino acid residues. In certain instances, linkers L1, L2 and L3 areeach independently GGSGGS (SEQ ID NO:95), GGSG (SEQ ID NO:96) or GGSGG(SEQ ID NO:97). In other instances, linkers L1 and L3 are GGSGGS (SEQ IDNO:95) and linker L2 is GGSG (SEQ ID NO:96) or GGSGG (SEQ ID NO:97).

In some embodiments, a bispecific tandem diabody has a sequence selectedfrom the group consisting of SEQ ID NOs:98-121. In other embodiments, abispecific tandem diabody is tandem diabody 01 (SEQ ID NO:98), 02 (SEQID NO:99), 03 (SEQ ID NO:100), 04 (SEQ ID NO:101), 05 (SEQ ID NO:102),06 (SEQ ID NO:103), 07 (SEQ ID NO:104), 08 (SEQ ID NO:105), 09 (SEQ IDNO:106), 10 (SEQ ID NO:107), 11 (SEQ ID NO:108), 12 (SEQ ID NO:109), 13(SEQ ID NO: 110), 14 (SEQ ID NO:111), 15 (SEQ ID NO:112), 16 (SEQ IDNO:113), 17 (SEQ ID NO:114), 18 (SEQ ID NO: 115), 19 (SEQ ID NO: 116),20 (SEQ ID NO: 117), 21 (SEQ ID NO: 118), 22 (SEQ ID NO: 119), 23 (SEQID NO: 120), or 24 (SEQ ID NO: 121).

In some embodiments, the bispecific, antigen-binding tandem diabodiespossess binding K_(D) of 10 nM or less to CD33 on CD33⁺ tumor cellsselected from HL-60, KG-1, and U-937.

In some embodiments, the bispecific, antigen-binding tandem diabodiesspecifically binds to an epitope of human CD33 which is within₆₂DQEVQEETQ₇₀ (SEQ ID NO:94) (amino acid residues 62-70 of SEQ ID NO:93)of human CD33.

In another aspect, provided herein are polynucleotides encoding a CD33binding protein or bispecific, tandem diabody of any of the aboveembodiments. In another aspect, provided herein are vectors comprisingthe described polynucleotides. In another aspect, provided herein arehost cells transformed with the described vectors.

In yet another aspect, provided herein are pharmaceutical compositionscomprising a CD33 binding protein or bispecific, tandem diabody of anyof the above embodiments and a pharmaceutically acceptable carrier.

In yet another aspect, provided herein methods of producing a CD33binding protein or bispecific, tandem diabody of any of the aboveembodiments comprising introducing into a host cell a polynucleotideencoding a CD33 binding protein or bispecific, tandem diabody of any ofthe above embodiments, or a vector comprising the describedpolynucleotides, culturing the host cell under conditions whereby theCD33 binding protein or the bispecific tandem diabody is expressed, andpurifying the expressed CD33 binding protein or the bispecific tandemdiabody.

Also provided herein are methods for the treatment of a CD33⁺ cancercomprising the administration of a bispecific, tandem diabody of any ofthe above embodiments to an individual suffering from CD33⁺ cancer. Insome embodiments, the CD33⁺ cancer is acute myeloid leukemia (AML),acute lymphoblastic leukemia (ALL), precursor B-cell lymphoblasticleukemia, myeloid sarcoma, multiple myeloma, acute lymphoma, acutelymphoblastic lymphoma or chronic myelomonocytic leukemia (CMML). Insome embodiments, the CD33⁺ cancer is acute myeloid leukemia (AML). Insome embodiments, the CD33⁺ cancer is multiple myeloma. In someembodiments, the CD33⁺ cancer is acute lymphoblastic leukemia (ALL).

Also provided herein are methods for the treatment of acute myeloidleukemia (AML) comprising the administration of a bispecific, tandemdiabody of any of the above embodiments to an individual suffering fromAML. In some embodiments, the AML is AML with Recurrent GeneticAbnormalities, AML with myelodysplasia-related changes, Therapy-relatedmyeloid neoplasms, Myeloid sarcoma, Myeloid proliferations related toDown syndrome, Blastic plasmacytoid dendritic cell neoplasm, or AML nototherwise categorized. In some embodiments, the AML is AML-M0, AML-M1,AML-M2, AML-M3, AML-M4, AML-M5, AML-M6, or AML-M7. In furtherembodiments, the AML is newly diagnosed, relapsed, or refractory.

Also provided herein are methods for the treatment of myeloid dysplasticsyndrome (MDS) comprising the administration of a bispecific, tandemdiabody of any of the above embodiments to an individual suffering fromMDS.

Also provided herein are methods for the treatment of myeloproliferativedisease (MPD) comprising the administration of a bispecific, tandemdiabody of any of the above embodiments to an individual suffering fromMPD.

Also provided herein are methods for the treatment of chronicmyelomonocytic leukemia (CMML) comprising the administration of abispecific, tandem diabody of any of the above embodiments to anindividual suffering from CMML.

Also provided herein are methods for the treatment of immune suppressionby myeloid derived suppressor cells (MDSCs) comprising theadministration of a bispecific, tandem diabody in any of the aboveembodiments to an individual suffering from immune suppression.

In the above methods for the treatment, in certain instances, themethods further comprise administering cytarabine, azacitidine,decitabine, an anthracycline (e.g., daunorubicin, idarubicin,doxorubicin, and the like), amsacrine, fludarabine, clofarabine,cladribine, nelarabine, methotrexate, bortezomib, carfilzomib,melphalan, ibrutinib, thalidomide, lenalidomide, pomalidomide,apremilast, an epipodophyllotoxin (e.g., etoposide, teniposide, and thelike), an anthracenedione (e.g., mitoxantrone, pixantrone, losoxantrone,piroxantrone, ametantrone and the like), an anti-CD20 agent (e.g.,rituximab, ocrelizumab, ofatumumab, and the like) or combinationsthereof.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 Schematic representation of the gene organization and a domainorder of CD3/CD33 tandem diabodies (TandAb®). Tandem diabodies areexpressed as a single polypeptide comprised of four variable domainsconnected via short peptide linkers L1, L2 and L3. Following expression,two monomeric polypeptides associate non-covalently head-to-tail to formthe functional homodimeric tandem diabody molecule. L1, L2, L3: Linker;V_(H): Heavy chain variable domain; V_(L): Light chain variable domain.

FIG. 2 CD3 engaging tandem diabody and its mode of action. Tandemdiabodies are tetravalent bispecific proteins that can engage cytotoxicT-cells via binding to CD3. The tandem diabody binds to a CD33⁺ tumorcell with two of four binding domains and to CD3 with the other twobinding domains. This T-cell/target cell binding (crosslinking) eventpromotes activation of the T-cell and promotes the subsequentdestruction of the tumor cell via ADCC.

FIG. 3 Domain order variants of CD33/CD3 tandem diabodies. Variations ofdomain order of variable heavy (VH) and variable light (VL) chainswithin gene sequences encoding tandem diabodies allows production ofantibodies with CD33 and CD3 specificities located on the inside oroutside of the molecule. Domain specificities, location of signalsequences (ss) and linkers (L1, L2, L3) and affinity tags (His) as wellas 5′- and 3′-ends are indicated.

FIG. 4 Comparison of positively enriched vs. negatively selected healthydonor T-cells. KG-1a cells were incubated with 10 pM (approx. 1 ng/mL)and 25 pM (approx. 2.5 ng/mL) of one of 10 selected tandem diabodies andeither negatively selected healthy donor T-cells or positively selectedhealthy donor T-cells at an E:T cell ratio of 1:1 or 3:1, as indicated.After 48 hours, cell counts were determined and cytotoxicity wasassessed with DAPI staining. Results are shown as mean±SEM for thepercentage of dead cells (upper panels) and the percentage of specificcytotoxicity (lower panels) from 3 independent experiments performed induplicate wells.

FIG. 5 Analysis strategy. Scatter and histogram plots from one healthydonor T-cell aliquot and 1 representative AML cell line (HL-60) andprimary AML specimen (AMP002) each illustrating the strategy pursued todetermine tandem diabody-induced cytotoxicity. FSC, forward scatter;SSC, side scatter.

FIG. 6 Screening cytotoxicity assays in CD33+ AML cell lines. ParentalHL-60 (A,B) and KG-1a (C,D) cells were incubated with 10 pM (approx. 1ng/mL) and 25 pM (approx. 2.5 ng/mL) of one of 22 CD33/CD3 tandemdiabody molecules or a non-binding control tandem diabody (00) andhealthy donor T-cells at an E:T cell ratio of either 1:1 (A,C) or 5:1(B,D) as indicated. After 48 hours, cell counts were determined andcytotoxicity was assessed with DAPI staining to quantify drug-specificcytotoxicity. Results are shown as mean±SEM for the percentage of DAPI⁺cells from 3 independent experiments performed in duplicate wells.Qualitatively similar results were obtained when cytotoxicity wasexpressed as the percentage of specific cytotoxicity.

FIG. 7 Selection of primary AML specimens for study. Frozen aliquotsfrom a total of primary human AML specimens were obtained for analysis.The percentage of AML blasts upon thaw was determined by flow cytometrybased on CD45/side-scatter properties. Viability of the specimens wasdetermined upon thaw as well after 48 hours in cytokine-containingliquid culture (without addition of tandem diabody molecules or healthydonor T-cells) via flow cytometry using DAPI as live/dead cell marker.Results for viability after thawing as well as after 48 hours aredepicted for all specimens, which had >58% AML blasts. Square: PrimaryAML specimens that showed a viability of >50% at thaw as well as >50%after 48 hours in cytokine-containing liquid culture which were includedin the final analyses.

FIG. 8 Tandem diabody-induced cytotoxicity in primary AML specimens.Primary AML specimens were incubated with 2.5 pM (approx. 250 pg/mL), 10pM (approx. 1 ng/mL), and 25 pM (approx. 2.5 ng/mL) of one of 9 tandemdiabody molecules without healthy donor T-cells added (A) or withhealthy donor T-cells at an E:T cell ratio of either 1:3 (B) or 1:1 (C)as indicated. After 48 hours, cell counts were determined andcytotoxicity was assessed with DAPI staining to quantify drug-specificcytotoxicity. Results are shown as mean±SEM for the percentage ofspecific cytotoxicity from experiments performed in duplicate wells.

FIG. 9 Amino acid sequences

(A) sequence of extracellular domain of human CD33 (aa 18-259) (SEQ IDNO: 93);

(B) complete sequence of tandem diabody 1 (SEQ ID NO:98);

(C) complete sequence of tandem diabody 2 (SEQ ID NO:99);

(D) complete sequence of tandem diabody 3 (SEQ ID NO: 100);

(E) complete sequence of tandem diabody 4 (SEQ ID NO: 101);

(F) complete sequence of tandem diabody 5 (SEQ ID NO:102);

(G) complete sequence of tandem diabody 6 (SEQ ID NO: 103);

(H) complete sequence of tandem diabody 7 (SEQ ID NO: 104);

(I) complete sequence of tandem diabody 8 (SEQ ID NO: 105);

(J) complete sequence of tandem diabody 9 (SEQ ID NO: 106);

(K) complete sequence of tandem diabody 10 (SEQ ID NO: 107);

(L) complete sequence of tandem diabody 11 (SEQ ID NO: 108);

(M) complete sequence of tandem diabody 12 (SEQ ID NO: 109);

(N) complete sequence of tandem diabody 13 (SEQ ID NO: 110);

(O) complete sequence of tandem diabody 14 (SEQ ID NO: 111);

(P) complete sequence of tandem diabody 15 (SEQ ID NO: 112);

(Q) complete sequence of tandem diabody 16 (SEQ ID NO: 113);

(R) complete sequence of tandem diabody 17 (SEQ ID NO: 114);

(S) complete sequence of tandem diabody 18 (SEQ ID NO: 115);

(T) complete sequence of tandem diabody 19 (SEQ ID NO: 116);

(U) complete sequence of tandem diabody 20 (SEQ ID NO: 117);

(B) complete sequence of tandem diabody 21 (SEQ ID NO: 118);

(W) complete sequence of tandem diabody 22 (SEQ ID NO: 119);

(X) complete sequence of tandem diabody 23 (SEQ ID NO: 120); and

(Y) complete sequence of tandem diabody 24 (SEQ ID NO:121). Underlinedsequences represent linkers L1, L2 and L3.

FIG. 10 Effect of tandem diabodies 16 and 12 on the growth of HL-60cells in NOD/scid mice. Eight experimental groups of immunodeficientNOD/scid mice were xenotransplanted by subcutaneous injection with asuspension of 4×10⁶ HL-60 cells on day 0. Prior to injection HL-60 cellswere mixed with 3×10⁶ purified T-cells from healthy donors. All animalsof the experimental groups transplanted with tumor cells and T-cellsreceived an intravenous bolus on days 0, 1, 2, 3 and 4 of either vehicle(control) or tandem diabody 16 or 12 at three different dose levels asindicated (0.1 μg, 1 μg, and 10 μg). One group without effector cellsand vehicle treatment served as an additional negative control.

FIG. 11 Anti-tumor activity of tandem diabody 16 in an AML XenograftModel. NOD/scid mice were sublethally irradiated (2 Gy) andsubcutaneously inoculated with 4×10⁶ HL-60 cells. On day 9 the animalsreceived a single bolus injection of anti-asialo GM1 rabbit Ab. Whentumors reached a volume between 50-150 mm³ (mean 73±11 mm³) on day 10animals were allocated to 3 treatment groups. Groups 2 and 3 (n=8) wereintraperitoneally injected with 1.5×10⁷ expanded and activated humanT-cells. From day 13 to day 21 (qdxd9) animals received either tandemdiabody 16 (Group 3) or vehicle into the lateral tail vein (Group 1 andGroup 2).

FIG. 12 Relative amount (A) and absolute counts (B) of human AML blastsin the bone marrow (BM) and spleen of NSG mice at day 38 after treatmentwith 5 μg (0.25 mg/kg) or 50 g (2.5 mg/kg) CD33/CD3 tandem diabody 12and 16.

FIG. 13 Kinetics of CD33/CD3 tandem diabody 16-mediated target celllysis. 1×10⁴ calcein-labeled HL-60 target cells were incubated withprimary human T-cells as effector cells at an E:T ratio of 25:1 in thepresence of serial dilutions of tandem diabody 16 or without antibody(w/o) for 30 min, 1 h, 2 h, 3 h, 4 h, or 5 h. At each time point, thefluorescent calcein released from lysed target cells was used tocalculated specific lysis. Mean and SD of three replicates are plotted.

FIG. 14 Kinetics of EC₅₀ and specific lysis values for CD33/CD3 tandemdiabody 16. EC₅₀ values (black solid circles) and tandem diabody16-mediated target cell lysis (open squares) were determined incalcein-release cytotoxicity assays at the indicated incubation times bynon-linear regression/sigmoidal dose-response and plotted.

FIG. 15 Cytotoxic activity in newly diagnosed, relapsed and refractoryAML patient samples.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect, described herein are binding proteinshaving specificity for at least CD33, preferably human CD33. In someembodiments, the CD33 binding proteins have specificity for human andcynomolgus CD33, i.e. are cross-reactive. In some embodiments, thesecross-reactive binding proteins bind to human and cynomolgous CD33 withsimilar affinity.

CD33 is expressed on myeloid cells, for example, such as the blasts ofacute myeloid leukemia (AML). For the isolation of antibody domainsspecific for CD33, such as human CD33, antibody libraries may bescreened. For example IgM phage display libraries can be screened byemploying, for example, a recombinant CD33-Fc fusion protein containingamino acids 1-243 of the extracellular domain of human CD33 (FIG. 9A,SEQ ID NO:93).

In some embodiments the CD33 binding protein has at least one CD33binding site comprising a light chain variable domain and a heavy chainvariable domain. The light chain variable domain comprises the lightchain CDR1, CDR2 and CDR3 and the heavy chain variable domain comprisesthe heavy chain CDR1, CDR2 and CDR3. In some embodiments these lightchain CDRs (CDR1, CDR2 and CDR3) are selected from the human CDRsequences shown in Table 1 (SEQ ID NOs:21-41). In certain instances, thelight chain CDR1 is selected from SEQ ID NOs:21-27. In certaininstances, the light chain CDR2 is selected from SEQ ID NOs:28-34. Incertain instances, the light chain CDR3 is selected from SEQ IDNOs:35-41.

In some embodiments these heavy chain CDRs (heavy chain CDR1, CDR2 andCDR3) are selected from the human CDR sequences shown in Table 2 (SEQ IDNOs:42-63). In certain instances, the heavy chain CDR1 is selected fromSEQ ID NOs:42-48. In certain instances, the heavy chain CDR2 is selectedfrom SEQ ID NOs:49-55. In certain instances, the heavy chain CDR3 isselected from SEQ ID NOs:56-63.

In some embodiments, the light and heavy CDRs are selected without thesurrounding framework sequences of the respective variable domains,which include framework sequences from other immunoglobulins orconsensus framework regions, optionally are further mutated and/orreplaced by other suitable framework sequences. Therefore providedherein in some embodiments, is a CD33 binding protein comprising a lightchain variable domain, wherein the light chain CDR1 is SEQ ID NO:21; thelight chain CDR2 is SEQ ID NO:28 and the light chain CDR3 is SEQ IDNO:35. In some embodiments, a CD33 binding protein comprises a lightchain variable domain, wherein the light chain CDR1 is SEQ ID NO:22; thelight chain CDR2 is SEQ ID NO:29 and the light chain CDR3 is SEQ IDNO:36. In some embodiments, a CD33 binding protein comprises a lightchain variable domain, wherein the light chain CDR1 is SEQ ID NO:23; thelight chain CDR2 is SEQ ID NO:30 and the light chain CDR3 is SEQ IDNO:37. In some embodiments, a CD33 binding protein comprises a lightchain variable domain, wherein the light chain CDR1 is SEQ ID NO:24; thelight chain CDR2 is SEQ ID NO:31 and the light chain CDR3 is SEQ IDNO:38. In some embodiments, a CD33 binding protein comprises a lightchain variable domain, wherein the light chain CDR1 is SEQ ID NO:25; thelight chain CDR2 is SEQ ID NO:32 and the light chain CDR3 is SEQ IDNO:39. In some embodiments, a CD33 binding protein comprises a lightchain variable domain, wherein the light chain CDR1 is SEQ ID NO:26; thelight chain CDR2 is SEQ ID NO:33 and the light chain CDR3 is SEQ IDNO:40. In some embodiments, a CD33 binding protein comprises a lightchain variable domain, wherein the light chain CDR1 is SEQ ID NO:27; thelight chain CDR2 is SEQ ID NO:34 and the light chain CDR3 is SEQ IDNO:41.

Also provided herein in some embodiments, is a CD33 binding proteincomprising a heavy chain variable domain, wherein the heavy chain CDR1is SEQ ID NO:42; the heavy chain CDR2 is SEQ ID NO:49 and the heavychain CDR3 is SEQ ID NO:56. In some embodiments, a CD33 binding proteincomprises a heavy chain variable domain, wherein the heavy chain CDR1 isSEQ ID NO:43; the heavy chain CDR2 is SEQ ID NO:50 and the heavy chainCDR3 is SEQ ID NO:57. In some embodiments, a CD33 binding proteincomprises a heavy chain variable domain, wherein the heavy chain CDR1 isSEQ ID NO:43; the heavy chain CDR2 is SEQ ID NO:50 and the heavy chainCDR3 is SEQ ID NO:58. In some embodiments, a CD33 binding proteincomprises a heavy chain variable domain, wherein the heavy chain CDR1 isSEQ ID NO:43; the heavy chain CDR2 is SEQ ID NO:50 and the heavy chainCDR3 is SEQ ID NO:59. In some embodiments, a CD33 binding proteincomprises a heavy chain variable domain, wherein the heavy chain CDR1 isSEQ ID NO:43; the heavy chain CDR2 is SEQ ID NO:50 and the heavy chainCDR3 is SEQ ID NO:60. In some embodiments, a CD33 binding proteincomprises a heavy chain variable domain, wherein the heavy chain CDR1 isSEQ ID NO:44; the heavy chain CDR2 is SEQ ID NO:51 and the heavy chainCDR3 is SEQ ID NO:61. In some embodiments, a CD33 binding proteincomprises a heavy chain variable domain, wherein the heavy chain CDR1 isSEQ ID NO:45; the heavy chain CDR2 is SEQ ID NO:52 and the heavy chainCDR3 is SEQ ID NO:62. In some embodiments, a CD33 binding proteincomprises a heavy chain variable domain, wherein the heavy chain CDR1 isSEQ ID NO:46; the heavy chain CDR2 is SEQ ID NO:53 and the heavy chainCDR3 is SEQ ID NO:63. In some embodiments, a CD33 binding proteincomprises a heavy chain variable domain, wherein the heavy chain CDR1 isSEQ ID NO:47; the heavy chain CDR2 is SEQ ID NO:54 and the heavy chainCDR3 is SEQ ID NO:63. In some embodiments, a CD33 binding proteincomprises a heavy chain variable domain, wherein the heavy chain CDR1 isSEQ ID NO:48; the heavy chain CDR2 is SEQ ID NO:55 and the heavy chainCDR3 is SEQ ID NO:63.

In further embodiments, a CD33 binding protein comprises a variablelight chain domain selected from amino acid sequences SEQ ID NOs:1-10shown in Table 3. In further embodiments, a CD33 binding proteincomprises a variable heavy chain domain selected from amino acidsequences SEQ ID NO: 11-20 shown in Table 4. In yet further embodiments,a CD33 binding protein comprises a variable light chain domain selectedfrom amino acid sequences SEQ ID NOs:1-10 shown in Table 3 and avariable heavy chain domain selected from amino acid sequences SEQ IDNO:11-20 shown in Table 4.

The term “binding protein” refers to an immunoglobulin derivative withantigen binding properties, i.e. immunoglobulin polypeptides orfragments thereof that contain an antigen binding site. The bindingprotein comprises variable domains of an antibody or fragments thereof.Each antigen-binding domain is formed by an antibody, i.e.immunoglobulin, variable heavy chain domain (VH) and an antibodyvariable light chain domain (VL) binding to the same epitope, whereasthe variable heavy chain domain (VH) comprises three heavy chaincomplementarity determining regions (CDR): CDR1, CDR2 and CDR3; and thevariable light chain domain (VL) comprises three light chaincomplementarity determining regions (CDR): CDR1, CDR2 and CDR3. In someinstances, the binding protein according to some embodiments herein isdevoid of immunoglobulin constant domains. In some instances, thevariable light and heavy chain domains forming the antigen binding siteis covalently linked with one another, e.g. by a peptide linker, or inother instances, the variable light and heavy chain domainsnon-covalently associate with one another to form the antigen bindingsite. The term “binding protein” refers also to antibody fragments orantibody derivatives including, for example, Fab, Fab′, F(ab′)₂, Fvfragments, single-chain Fv, tandem single-chain Fv ((scFv)₂, Bi-specificT-cell engagers (BiTE®), dual affinity retargeting antibodies (DART™),diabody and tandem diabody (TandAb®). Furthermore, in certain instances,the binding protein is multivalent, i.e. has two, three or more bindingsites for CD33.

TABLE 1 Amino acid sequences of anti-CD33 variable light chain CDR1, CDR2 and CDR3 Sequence Light Chain CDR identifierCDR Sequence CDR1 SEQ ID NO: 21 GGNNIGSTTVH SEQ ID NO: 22 SGSRSNIGSNTVNSEQ ID NO: 23 SGSSSNIGSNTVN SEQ ID NO: 24 TGSSSNIGAGYDVH SEQ ID NO: 25SGSSSNIGSNIVN SEQ ID NO: 26 SGSSSNIGSNTVK SEQ ID NO: 27 SGSSSNIGDNVVNCDR2 SEQ ID NO: 28 DDNERPS SEQ ID NO: 29 GNNQRPS SEQ ID NO: 30 SDNQRPSSEQ ID NO: 31 GNSNRPS SEQ ID NO: 32 SNNQRPS SEQ ID NO: 33 SNNQRSSSEQ ID NO: 34 STNKRPS CDR3 SEQ ID NO: 35 QVWDSGSDH SEQ ID NO: 36ATWDDSLIG SEQ ID NO: 37 ATWDDSLNG SEQ ID NO: 38 QSYDSSLSD SEQ ID NO: 39AAWDDSLKG SEQ ID NO: 40 AAWDDSLNG SEQ ID NO: 41 AAWDDSLSA

TABLE 2 Amino acid sequences of anti-CD33 variable heavychain CDR1, CDR2 and CDR3 Sequence Heavy Chain CDR identifierCDR Sequence CDR1 SEQ ID NO: 42 SNYGIH SEQ ID NO: 43 TSYDINSEQ ID NO: 44 TSYYMH SEQ ID NO: 45 TSYWIG SEQ ID NO: 46 SSYAISSEQ ID NO: 47 SSYGIS SEQ ID NO: 48 DSYAIS CDR2 SEQ ID NO: 49LISYDGNKKFYADSVKG SEQ ID NO: 50 WMNPNSGNTGFAQKFQG SEQ ID NO: 51GIINPSGGSTSYAQKFQG SEQ ID NO: 52 IIYPGDSDTRYSPSFQG SEQ ID NO: 53GIYPIFGSANYAQKFQG SEQ ID NO: 54 GIIPIFGSAHYAQKFQG SEQ ID NO: 55GIIPIFGSAHYSQKFQG CDR3 SEQ ID NO: 56 DRLESAAFDY SEQ ID NO: 57DRANTDFSYGMDV SEQ ID NO: 58 DRAVTDYYYGMDV SEQ ID NO: 59 DRANTDYSFGMDVSEQ ID NO: 60 DRANTDYSLGMDV SEQ ID NO: 61 DVVPAAIDYYGMDV SEQ ID NO: 62HKRGSDAFDI SEQ ID NO: 63 EYYYDSSEWAFDI

TABLE 3Amino acid sequences of all anti-CD33 variable light chain domains (amino acidsequences of variable light chain CDRL CDR2 and CDR3 are in bold and underlined)anti-CD33 Sequence clone identifierVariable light chain (VL) domain Sequence 01 SEQ ID NO: 1SYELTQPPSVSVAPGQTAMITC GGNNIGSTTVH WYQQKPGQAPVLVV Y DDNERPSGIPERFSGSNSGSTATLTINRVEAGDEADYYC QVWDSGSD H VVFGGGTKLTVL 02 SEQ ID NO: 2QSVLTQPPSASGTPGQRVTISC SGSRSNIGSNTVN WYQQLPGTAPKLLI Y GNNQRPSGVPDRFSGSKSGSSASLAISGLQSEDEADYYC ATWDDSLI G WVFGGGTKLTVL 03 SEQ ID NO: 3QSVLTQPPSASGTPGQRVTISC SGSRSNIGSNTVN WYQQLPGTAPKLLI Y GNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYC ATWDDSLI G WVFGGGTKLTVL 04 SEQ ID NO: 4QSVLTQPPSASGTPGQRVTISC SGSRSNIGSNTVN WYQQLPGTAPKLLI Y GNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYC ATWDDSLI G WVFGGGTKLTVL 05 SEQ ID NO: 5QSVLTQPPSASGTPGQRVTISC SGSRSNIGSNTVN WYQQLPGTAPKLLI Y GNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYC ATWDDSLI G WVFGGGTKLTVL 06 SEQ ID NO: 6QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNTVN WYQQLPGTAPKLLI Y SDNQRPSGVPDRFSGSKSGSSASLAISGLQSDDEADYYCATWDDSLN GAVFGGGTKLTVL 07 SEQ ID NO: 7QSVLTQPPSVSGAPGQRVTISC TGSSSNIGAGYDVH WYQQLPGTAPKL LIY GNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYC QSYDSSL SD VVFGGGTKLTVL 08 SEQ ID NO: 8QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNIVN WYQQLPGTAPKLLIY SNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYC AAWDDSLKG YVFGGGTKLTVL 09 SEQ ID NO: 9QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNTVK WYQQLPGTAPKLLI Y SNNQRSSGVPDRFSGSKSGSSASLAISGLQSEDEADYYC AAWDDSLN G YVFGGGTKLTVL 10SEQ ID NO: 10 QSVLTQPPSASGTPGQRVTISC SGSSSNIGDNVVN WYQQLPGTAPKLLI YSTNKRPS GVPDRFSGSKSGSSASLAISGLQSEDEADYYC AAWDDSLS A YVFGGGTKLTVL

TABLE 4Amino acid sequence of anti-CD33 variable heavy chain domain (amino acidsequences of variable heavy chain CDR1, CDR2 and CDR3 are in bold and underlined)anti-CD33 Sequence clone identifierVariable heavy chain (VH) domain Sequence 01 SEQ ID NO: 11QVQLQESGGGVVQPGRSLRLSCAASGFSF SNYGIH WVRQAPGKGLEWVA LISYDGNKKFYADSVKGRFAISRDTSKNTVDLQMTSLRPEDTAVYYCAK DRLESAAFDY WGQGTLVTVSS 02 SEQ ID NO: 12QVQLVQSGAEVKKPGASVKVSCKASGYTF TSYDIN WVRQAPGQGLEWM G WMNPNSGNTGFAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC AR DRANTDFSYGMDV WGQGTLVTVSS 03SEQ ID NO: 13 QVQLVQSGAEVKKPGASVKVSCKASGYTF TSYDIN WVRQAPGQGLEWM GWMNPNSGNTGFAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYC AR DRAVTDYYYGMDVWGQGTLVTVSS 04 SEQ ID NO: 14 QVQLVQSGAEVKKPGASVKVSCKASGYTF TSYDINWVRQAPGQGLEWM G WMNPNSGNTGFAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYC ARDRANTDYSFGMDV WGQGTLVTVSS 05 SEQ ID NO: 15 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDIN WVRQAPGQGLEWM G WMNPNSGNTGFAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR DRANTDYSLGMDV WGQGTLVTVSS 06 SEQ ID NO: 16QVQLVQSGAEVKKPGASVKVSCKASGYTF TSYYMH WVRQAPGQGLEW M GIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC AR DVVPAAIDYYGMDV WGQGTTVTVSS 07SEQ ID NO: 17 QVQLVQSGAEVKKPGESLKISCKGSGYSF TSYWIG WVRQMPGKGLEWMGIIYPGDSDTRYSPSFQG QVTISADKSISTAYLQWSSLKASDTAMYYCAR HKRGSDAFDIWGQGTTVTVSS 08 SEQ ID NO: 18 QVQLVQSGAEVKKPGSSVKVSCKASGGTF SSYAISWVRQAPGQGLEWMG GIYPIFGSANYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR EYYYDSSEWAFDI WGQGTLVTVSS 09 SEQ ID NO: 19 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGIS WVRQAPGQGLEWM G GIIPIFGSAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR EYYYDSSEWAFDI WGQGTLVTVSS 10SEQ ID NO: 20 QVQLVQSGAEVKKPGSSVKVSCKASGGTF DSYAIS WVRQAPGQGLEWM GGIIPIFGSAHYSQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAREYYYDSSEWAFDIWGQGTLVTVSS

In some embodiments, a binding protein conferring specificity to CD33 isselected from one of the following combinations of a variable heavychain domain and a variable light chain domain forming the human CD33binding site shown in Table 3 and in Table 4. Non-limiting examplesinclude (i) SEQ ID NO: 1 and SEQ ID NO: 11, (ii) SEQ ID NO:2 and SEQ IDNO: 12, (iii) SEQ ID NO:3 and SEQ ID NO: 13, (iv) SEQ ID NO:4 and SEQ IDNO: 14, (v) SEQ ID NO:5 and SEQ ID NO:15, (vi) SEQ ID NO:6 and SEQ IDNO:16, (vii) SEQ ID NO:7 and SEQ ID NO:17, (viii) SEQ ID NO:8 and SEQ IDNO:18, (ix) SEQ ID NO:9 and SEQ ID NO:19, and (x) SEQ ID NO:10 and SEQID NO:20.

Also described herein are binding proteins that not only havespecificity for CD33, but which also have at least one furtherfunctional domain. In a further embodiment at least one furtherfunctional domain is an effector domain. An “effector domain” comprisesa binding site of an antibody specific for an effector cell, which canstimulate or trigger cytotoxicity, phagocytosis, antigen presentation,cytokine release. Such effector cells are, for example, but not limitedto, T-cells. In particular, the effector domain comprises at least oneantibody variable heavy chain domain and at least one variable lightchain domain forming an antigen binding site for an antigen on T-cells,such as, for example, human CD3.

Thus, in some embodiments, the CD33 binding protein is multifunctional.The term multifunctional as used herein means that a binding proteinexhibits two or more different biological functions. For example, thedifferent biological functions are different specificities for differentantigens. In certain instances, the multifunctional CD33 binding proteinis multispecific, i.e. has binding specificity to CD33 and one or morefurther antigens. In certain instances, the binding protein isbispecific with specificities for CD33 and CD3. Such bispecific bindingproteins include, for example, bispecific monoclonal antibodies of theclasses IgA, IgD, IgE, IgG or IgM, diabodies, single-chain diabodies(scDb), tandem single chain Fv (scFv)2, for example Bi-specific T-cellengagers (BiTE®), dual affinity retargeting antibodies (DART™), tandemdiabodies (TandAb®), and flexibodies.

In certain embodiments, the CD3 binding site of a bispecific CD33 andCD3 binding protein has specificity for human CD3 and, in someinstances, cynomolgus CD3. Examples of such a binding site arepolypeptides comprising the VH domain CDR1, CDR2 and CDR3 from thesequences shown in Table 5 (SEQ ID NOs:64-67) and VL domain CDR1, CDR2and CDR3 from the sequence shown in Table 6 (SEQ ID NOs:68-71). Incertain instances, a CD3 binding site is the combination of the variableheavy chain domain of SEQ ID NO:64 and the variable light chain domainof SEQ ID NO:68. In certain instances, a CD3 binding site is thecombination of the variable heavy chain domain of SEQ ID NO:65 and thevariable light chain domain of SEQ ID NO:69. In certain instances, a CD3binding site is the combination of the variable heavy chain domain ofSEQ ID NO:66 and the variable light chain domain of SEQ ID NO:70. Incertain instances, a CD3 binding site is the combination of the variableheavy chain domain of SEQ ID NO:67 and the variable light chain domainof SEQ ID NO:71.

TABLE 5Amino acid sequence of an anti-CD3 variable heavy chain domain (amino acidsequences of variable heavy chain CDR1, CDR2 and CDR3 are in bold and underlined)anti-CD3 VH domain Sequence SEQ ID NO: 64 EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMN WVRQAPGKGLEWVG RIRSKYNNY CD3-01 ATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR HGNFGNSYVSYFAY WG QGTLVTVSSSEQ ID NO: 65 EVQLVESGGGLVQPGGSLRLSCAASGFTF STYAMN WVRQAPGKGLEWVGRIRSKYNNY CD3-02 ATYYADSVKD RFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAY WG QGTLVTVSS SEQ ID NO: 66 EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMN WVRQAPGKGLEWVG RIRSKYNNY CD3-03 ATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR HGNFGNSYVSWFAY WG QGTLVTVSSSEQ ID NO: 67 EVQLVESGGGLVQPGGSLRLSCAASGFTF STYAMN WVRQAPGKGLEWVGRIRSKYNNY CD3-04 ATYYADSVKD RFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAY WG QGTLVTVSS

TABLE 6Amino acid sequence of an anti-CD3 variable light chain domain (amino acidsequences of variable light chain CDR1, CDR2 and CDR3 are in bold and underlined)anti-CD3 VL domain Sequence SEQ ID NO: 68 DIQMTQSPSSLSASVGDRVTITCRSSTGAVTTSNYAN WVQQKPGKAPKALIG GTNKRAP CD3-01GVPSRFSGSLIGDKATLTISSLQPEDFATYYC ALWYSNL WVFGQGTKVEIK SEQ ID NO: 69DIQMTQSPSSLSASVGDRVTITC RSSTGAVTTSNYAN WVQQKPGKAPKGLIG GTNKRAP CD3-02GVPARFSGSGSGTDFTLTISSLQPEDFATYYC ALWYSNL WVFGQGTKVEIK SEQ ID NO: 70DIQMTQSPSSLSASVGDRVTITC RSSTGAVTTSNYAN WVQQKPGKAPKGLIG GTNKRAP CD3-03GVPSRFSGSLIGDKATLTISSLQPEDFATYYC ALWYSNL WVFGQGTKVEIK SEQ ID NO: 71DIQMTQSPSSLSASVGDRVTITC RSSTGAVTTSNYAN WVQQKPGKAPKGLIG GTNKRAP CD3-04GVPSRFSGSLIGTDFTLTISSLQPEDFATYYC ALWYSNL WVFGQGTKVEIK

In further embodiments, the CD3 binding site of a bispecific CD33 andCD3 binding protein has a variable heavy chain domain comprising a CDR1sequence of STYAMN (SEQ ID NO:72). In further embodiments, the CD3binding site of a bispecific CD33 and CD3 binding protein has a variableheavy chain domain comprising a CDR2 sequence of RIRSKYNNYATYYADSVKD(SEQ ID NO:73). In further embodiments, the CD3 binding site of abispecific CD33 and CD3 binding protein has a variable heavy chaindomain comprising a CDR3 sequence of HGNFGNSYVSWFAY (SEQ ID NO:74). Infurther embodiments, the CD3 binding site of a bispecific CD33 and CD3binding protein has a variable heavy chain domain comprising a CDR3sequence of HGNFGNSYVSYFAY (SEQ ID NO:75). In yet further embodiments,the CD3 binding site has a variable heavy chain domain comprising aCDR1, CDR2 and CDR3 sequence of SEQ ID NOs:72-74 respectively. In yetfurther embodiments, the CD3 binding site has a variable heavy chaindomain comprising a CDR1, CDR2 and CDR3 sequence of SEQ ID NOs:72, 73and 75 respectively.

In further embodiments, the CD3 binding site of a bispecific CD33 andCD3 binding protein has a variable heavy chain domain comprising a CDR1sequence selected from the group consisting of NTYAMN (SEQ ID NO:76),NTYAMH (SEQ ID NO:77) and NKYAMN (SEQ ID NO:78). In further embodiments,the CD3 binding site of a bispecific CD33 and CD3 binding protein has avariable heavy chain domain comprising a CDR2 sequence selected from thegroup consisting of RIRNKYNNYATYYADSVKD (SEQ ID NO:79),RIRNKYNNYATEYADSVKD (SEQ ID NO:80), RIRSKYNNYATEYAASVKD (SEQ ID NO:81),RIRNKYNNYATEYAASVKD (SEQ ID NO:82), RIRSKYNNYATYYADSVKG (SEQ ID NO:83)and RIRSKYNNYATEYADSVKS (SEQ ID NO:84). In further embodiments, the CD3binding site of a bispecific CD33 and CD3 binding protein has a variableheavy chain domain comprising a CDR3 sequence selected from the groupconsisting of HGNFGDSYVSWFAY (SEQ ID NO:85), HGNFGNTYVSWFAY (SEQ IDNO:86), HGNFGCSYVSWFAY (SEQ ID NO:87), HGNFGNSYISYWAY (SEQ ID NO:88) andHGNFGNSYVSFFAY (SEQ ID NO:89).

In yet further embodiments, the CD3 binding site has a variable heavychain domain comprising a CDR1, CDR2 and CDR3 sequence of SEQ ID NOs:76,73 and 74 respectively, SEQ ID NOs:76, 79 and 74 respectively, SEQ IDNOs:76, 80 and 74 respectively, SEQ ID NOs:76, 81 and 74 respectively,SEQ ID NOs:76, 82 and 74 respectively, SEQ ID NOs:76, 83 and 74respectively, SEQ ID NOs:72, 83 and 74 respectively, SEQ ID NOs:72, 83and 85 respectively, SEQ ID NOs:76, 83 and 86 respectively, SEQ IDNOs:77, 83 and 74 respectively, SEQ ID NOs:72, 83 and 87 respectively,SEQ ID NOs:78, 73 and 88 respectively or SEQ ID NOs:78, 84 and 89respectively.

In further embodiments, the CD3 binding site of a bispecific CD33 andCD3 binding protein has a variable light chain domain comprising a CDR1sequence ofRSSTGAVTTSNYAN (SEQ ID NO:90). In further embodiments, theCD3 binding site of a bispecific CD33 and CD3 binding protein has avariable light chain domain comprising a CDR2 sequence of GTNKRAP (SEQID NO:91). In further embodiments, the CD3 binding site of a bispecificCD33 and CD3 binding protein has a variable light chain domaincomprising a CDR3 sequence of ALWYSNL (SEQ ID NO:92). In yet furtherembodiments, the CD3 binding site has a variable light chain domaincomprising a CDR1, CD2 and CD3 sequence of SEQ ID NOs:90-92respectively.

In certain instances, the CD3 binding site has a high affinity to CD3.Alternatively, in other instances, the CDR1, CDR2, CDR3 from theheavy-chain domain as well as the light-chain domain or, optionally, thevariable light-chain domains and variable heavy-chain domains is derivedfrom other CD3 antibodies, such as, for example UCHT1, muromonab-CD3(OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion),and the like.

In another aspect, described herein are CD33 binding proteins as well asthe bispecific CD33 and CD3 binding proteins that are humanized or fullyhuman, i.e. of human origin.

In some embodiments, a bispecific CD33 and CD3 binding protein has oneof the following combinations providing CD33 and CD3 specificity byvariable light and heavy chain domains for CD33 and CD3: include, butare not limited to, (i) SEQ ID NOs:2, 12, 65 and 69, (ii) SEQ ID NOs:3,13, 65 and 69, (iii) SEQ ID NOs:4, 14, 65 and 69, (iv) SEQ ID NOs:5, 15,65 and 69, (v) SEQ ID NOs: 1, 11, 64 and 68, (vi) SEQ ID NOs:2, 12, 64and 68, (vii) SEQ ID NOs:2, 12, 66 and 70, (viii) SEQ ID NOs:4, 14, 66and 70, (ix) SEQ ID NOs:5, 15, 66 and 70, and (x) SEQ ID NOs:3, 13, 64and 68, (xi) SEQ ID NOs:3, 13, 67 and 71, (xii) SEQ ID NOs:4, 14, 64 and68, (xiii) SEQ ID NOs:5, 15, 64 and 68, (xiv) SEQ ID NOs:7, 17, 64 and68, (xv) SEQ ID NOs:6, 16, 64 and 68, (xvi) SEQ ID NOs:6, 16, 67 and 71,(xvii) SEQ ID NOs:8, 18, 64 and 68, (xviii) SEQ ID NOs:9, 19, 64 and 68;(xix) SEQ ID NOs:9, 19, 67 and 71, and (xx) SEQ ID NOs: 10, 20, 64 and68.

Conserved Variants of CDR Sequences and Heavy and Light Chain Domains

In alternative embodiments, the heavy and light chain domainsincorporate immunologically active homologues or variants of the CDRsequences described herein. Accordingly in some embodiments, a CDRsequence in a heavy or light chain domain that binds to CD33 or CD3 issimilar to, but not identical to, the amino acid sequence depicted inSEQ ID NOs: 21-63 or 72-92. In certain instances, a CDR variant sequencehas a sequence identity of 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%,90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% compared to thesequence of SEQ ID NOs: 21-63 or 72-90 and which is immunologicallyactive.

In further instances, a CDR variant sequence incorporates 1, 2, 3, 4, or5 conserved amino acid substitutions. Conservative substitutions includeamino acid substitutions that substitute a given amino acid with anotheramino acid of similar characteristics and further include, among thealiphatic amino acids interchange of alanine, valine, leucine, andisoleucine; interchange of the hydroxyl residues serine and threonine,exchange of the acidic residues aspartate and glutamate, substitutionbetween the amide residues asparagine and glutamine, exchange of thebasic residues lysine and arginine, and replacements among the aromaticresidues phenylalanine and tyrosine.

In yet further instances, a CDR variant sequence incorporatessubstitutions that enhance properties of the CDR such as increase instability, resistance to proteases and/or binding affinities to CD33 orCD3.

In other instances, a CDR variant sequence is modified to changenon-critical residues or residues in non-critical regions. Amino acidsthat are not critical can be identified by known methods, such asaffinity maturation, CDR walking, site-directed mutagenesis,crystallization, nuclear magnetic resonance, photoaffinity labeling, oralanine-scanning mutagenesis.

In further alternative embodiments, the CD33 and CD3 binding proteinscomprise heavy and light chain domains that are immunologically activehomologues or variants of heavy and light chain domain sequencesprovided herein. Accordingly, in some embodiments, a CD33 and CD3binding protein comprises a heavy or light chain domain sequence that issimilar to, but not identical to, the amino acid sequence depicted inSEQ ID NOs: 1-20 or 64-71. In certain instances, a variant heavy orlight chain domain sequence has a sequence identity of 99%, 98%, 97%,96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%,82%, 81%, or 80% compared to the sequence of SEQ ID NOs: 1-20 or 64-71and which is immunologically active.

In further instances, a variant heavy or light chain domain sequenceincorporates 1, 2, 3, 4, or 5 conserved amino acid substitutions.Conservative substitutions include amino acid substitutions thatsubstitute a given amino acid with another amino acid of similarcharacteristics and further include, among the aliphatic amino acidsinterchange of alanine, valine, leucine, and isoleucine; interchange ofthe hydroxyl residues serine and threonine, exchange of the acidicresidues aspartate and glutamate, substitution between the amideresidues asparagine and glutamine, exchange of the basic residues lysineand arginine, and replacements among the aromatic residues phenylalanineand tyrosine.

In yet further instances, a variant heavy or light chain domain sequenceincorporates substitutions that enhance properties of the CDR such asincrease in stability, resistance to proteases and/or binding affinitiesto CD33 or CD3.

In other instances, a variant heavy or light chain domain sequence ismodified to change non-critical residues or residues in non-criticalregions. Amino acids that are not critical can be identified by knownmethods, such as affinity maturation, CDR walking, site-directedmutagenesis, crystallization, nuclear magnetic resonance, photoaffinitylabeling, or alanine-scanning mutagenesis.

CD33 and CD3 Bispecific and Tandem Diabodies

In another aspect, a CD33 binding protein or the bispecific CD33 and CD3binding protein is a dimer, i.e. comprises two polypeptides with antigenbinding sites for CD33 and CD3.

Also provided herein in another aspect, is a dimeric and bispecific CD33and CD3 binding protein in the format of a tandem diabody (TandAb®).Such tandem diabodies are constructed by linking four antibody variablebinding domains (two heavy-chain variable domains (VH) and twolight-chain variable domains (VL) in a single gene construct (FIG. 1)enabling homo-dimerization. In such tandem diabodies the linker lengthis such that it prevents intramolecular pairing of the variable domainsso that the molecule cannot fold back upon itself to form a single-chaindiabody, but rather is forced to pair with the complementary domains ofanother chain. The domains are also arranged such that the correspondingVH and VL domains pair during this dimerization. Following expressionfrom a single gene construct, two identical polypeptide chains foldhead-to-tail forming a functional non-covalent homodimer ofapproximately 105 kDa (FIG. 1). Despite the absence of intermolecularcovalent bonds, the homodimer is highly stable once formed, remainsintact and does not revert back to the monomeric form.

Tandem diabodies have a number of properties that provide advantagesover traditional monoclonal antibodies and other smaller bispecificmolecules. Tandem diabodies contain only antibody variable domains andtherefore are contemplated to lack side effects or non-specificinteractions that may be associated with an Fc moiety. For example, Fcreceptors which can bind to Fc domains are found on numerous cell typessuch as white blood cells (e.g., basophils, B-cells, eosinophils,natural killer cells, neutrophils and the like) or Kuppfer cells.Because tandem diabodies allow for bivalent binding to each of CD33 andCD3, the avidity is the same as that of an IgG. The size of a tandemdiabody, at approximately 105 kDa, is smaller than that of an IgG, whichmay allow for enhanced tumor penetration. However, this size is wellabove the renal threshold for first-pass clearance, offering apharmacokinetic advantage compared with smaller bispecific formats basedon antibody-binding domains or non-antibody scaffolds. Moreover tandemdiabodies are advantageous over other bispecific binding proteins suchas BiTE or DART molecules based on this pharmacokinetic and avidityproperties resulting in longer intrinsic half-lives and rapidcytotoxicity. Tandem diabodies are well expressed in host cells, forexample, mammalian CHO cells. It is contemplated that robust upstreamand downstream manufacturing process is available for tandem diabodies.

The CD33 and CD3 bispecific tandem diabodies described herein aredesigned to allow specific targeting of CD33⁺ tumor cells by recruitingcytotoxic T-cells. This improves ADCC (antibody dependent cell-mediatedcytotoxicity) as compared to full length antibodies directed to a soleantigen and are not capable of directly recruiting cytotoxic T-cells. Incontrast, by engaging CD3 molecules expressed specifically on thesecells, the tandem diabody can crosslink cytotoxic T-cells with CD33⁺tumor cells in a highly specific fashion, thereby significantlyincreasing the cytotoxic potential of such molecules. This mechanism isoutlined in FIG. 2. The tandem diabody displays strong, specific andefficient ADCC. It is reported that T-cells can play a role incontrolling tumor growth. For example, the presence of cytotoxic T-cellsin colorectal tumors as well as lymph nodes from NHL patients was shownto correlate with a better clinical outcome. Furthermore, the potentialof therapies designed to induce T-cell responses has been demonstratedfor melanoma vaccines, as well as the antibody directed against CTLA-4,a negative regulator of T-cell activation. The tandem diabodiesdescribed herein engage cytotoxic T-cells via binding to thesurface-expressed CD3, which forms part of the T-cell receptor.Simultaneous binding of this tandem diabody to CD3 and to CD33 expressedon the surface of particular tumor cells causes T-cell activation andmediates the subsequent lysis of the tumor cell (FIG. 2).

Therefore, in a further aspect is a multispecific, tandem diabody. Insome embodiments, a multispecific tandem diabody has specificities totwo, three or more different epitopes, wherein two or more epitopes canbe of the same antigen target or of different antigen targets. Incertain embodiments the multispecific, tandem diabody is bispecific andtetravalent, i.e. comprises four antigen-binding sites. Such abispecific tandem diabody binds with at least one antigen-binding site,to human CD3 and to human CD33, wherein in certain instances, the tandemdiabody binds with two antigen-binding sites to human CD3 and with twoother antigen-binding sites to human CD33, i.e. the tandem diabody bindsbivalently to each antigen.

In some embodiments, a bispecific, antigen-binding tandem diabody isspecific to human CD33 and human CD3, wherein said tandem diabodycomprises a first polypeptide and a second polypeptide, each polypeptidehaving at least four variable chain domains linked one after another,wherein each polypeptide comprises

(i) a variable heavy chain (VH) domain specific to human CD33;

(ii) a variable light chain (VL) domain specific to human CD33;

(iii) a VH domain specific for human CD3, and

(iv) a VL domain specific for human CD3.

In particular embodiments, a bispecific tandem diabody specificallybinds to an epitope of human CD33 which is within ₆₂DQEVQEETQ₇₀ (SEQ IDNO:94) (amino acid residues 62-70 of SEQ ID NO:93) of human CD33. Inparticular instances, such a tandem diabody comprises a firstpolypeptide and a second polypeptide, each polypeptide having at leastfour variable chain domains linked one after another, wherein eachpolypeptide comprises

-   -   (i) a variable heavy chain domain specific to an epitope of        human CD33 which is within ₆₂DQEVQEETQ₇₀ (SEQ ID NO:94) (amino        acid residues 62-70 of SEQ ID NO:93) of human CD33;    -   (ii) a variable light chain domain specific to an epitope of        human CD33 which is within ₆₂DQEVQEETQ₇₀ (SEQ ID NO:94) (amino        acid residues 62-70 of SEQ ID NO:93) of human CD33;    -   (iii) a variable heavy chain domain specific for human CD3, and    -   (iv) a variable light chain domain specific for human CD3.

In other embodiments, described herein are CD33/CD3 tandem diabodiesthat have an affinity to CD33 on CD33⁺ cells with a K_(D) of 10 nM orless, 5 nM or less, 1 nM or less, or 0.5 nM or less. The CD33⁺ cells canbe selected from tumor cells such as, for example, HL-60 or KG-1.

In a further embodiment a CD33/CD3 tandem diabody described herein bindsCD3 and in certain instances, the epsilon chain of CD3 on CD3⁺ cells,particularly T-cells, with a K_(D) of 10 nM or less, 5 nM or less or 2nM or less.

In some embodiments, each polypeptide of a bispecific tandem diabodycomprises one of the following combinations of the four variable chaindomains: (i) SEQ ID NOs:2, 12, 65 and 69, (ii) SEQ ID NOs:3, 13, 65 and69, (iii) SEQ ID NOs:4, 14, 65 and 69, (iv) SEQ ID NOs:5, 15, 65 and 69,(v) SEQ ID NOs: 1, 11, 64 and 68, (vi) SEQ ID NOs:2, 12, 64 and 68,(vii) SEQ ID NOs:2, 12, 66 and 70, (viii) SEQ ID NOs:4, 14, 66 and 70,(ix) SEQ ID NOs:5, 15, 66 and 70, and (x) SEQ ID NOs:3, 13, 64 and 68,(xi) SEQ ID NOs:3, 13, 67 and 71, (xii) SEQ ID NOs:4, 14, 64 and 68,(xiii) SEQ ID NOs:5, 15, 64 and 68, (xiv) SEQ ID NOs:7, 17, 64 and 68,(xv) SEQ ID NOs:6, 16, 64 and 68, (xvi) SEQ ID NOs:6, 16, 67 and 71,(xvii) SEQ ID NOs:8, 18, 64 and 68, (xviii) SEQ ID NOs:9, 19, 64 and 68;(xix) SEQ ID NOs:9, 19, 67 and 71, and (xx) SEQ ID NOs:10, 20, 64 and68.

As used herein, “dimer” refers to a complex of two polypeptides. Incertain embodiments, the two polypeptides are non-covalently associatedwith each other, in particular with the proviso that there is nocovalent bond between the two polypeptides. In certain instances, thetwo polypeptides have covalent associations such as disulfide bonds thatform to aid in stabilization of the dimer. In certain embodiments, thedimer is homodimeric, i.e. comprises two identical polypeptides. Theterm “polypeptide” refers to a polymer of amino acid residues linked byamide bonds. The polypeptide is, in certain instances, a single chainfusion protein, which is not branched. In the polypeptide the variableantibody domains are linked one after another. The polypeptide, in otherinstances, may have contiguous amino acid residues in addition to thevariable domain N-terminal and/or C-terminal residues. For example, suchcontiguous amino acid residues may comprise a Tag sequence, in someinstances at the C-terminus, which is contemplated to be useful for thepurification and detection of the polypeptide.

In one aspect, each polypeptide of the bispecific tandem diabodycomprises four variable domains, a variable light chain (VL) and avariable heavy chain (VH) of a CD3 binding protein as well as a variablelight chain (VL) and a variable heavy chain (VH) of a CD33 bindingprotein. In certain embodiments, four variable domains are linked bypeptide linkers L1, L2 and L3 and in some instances arranged from the N-to the C-terminus as follows:

Domain Order: (1) VL(CD3)-L1-VH(CD33)-L2-VL(CD33)-L3-VH(CD3); or (2)VH(CD3)-L1-VL(CD33)-L2-VH(CD33)-L3-VL(CD3); or (3)VL(CD33)-L1-VH(CD3)-L2-VL(CD3)-L3-VH(CD33); or (4)VH(CD33)-L1-VL(CD3)-L2-VH(CD3)-L3-VL(CD33).

The length of the linkers influences the flexibility of theantigen-binding tandem diabody according to reported studies.Accordingly, in some embodiments, the length of the peptide linkers L1,L2 and L3 is such that the domains of one polypeptide can associateintermolecularly with the domains of another polypeptide to form thedimeric antigen-binding tandem diabody. In certain embodiments, suchlinkers are “short”, i.e. consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11 or 12 amino acid residues. Thus, in certain instances, the linkersconsist of about 12 or less amino acid residues. In the case of 0 aminoacid residues, the linker is a peptide bond. Such short linkers favorthe intermolecular dimerization of the two polypeptides by binding andforming correct antigen-binding sites between antibody variable lightchain domains and antibody variable heavy chain domains of differentpolypeptides. Shortening the linker to about 12 or less amino acidresidues generally prevents adjacent domains of the same polypeptidechain from intramolecular interaction with each other. In someembodiments, these linkers consist of about 3 to about 10, for example4, 5 or 6 contiguous amino acid residues.

Regarding the amino acid composition of the linkers, peptides areselected that do not interfere with the dimerization of the twopolypeptides. For example, linkers comprising glycine and serineresidues generally provide protease resistance. The amino acid sequenceof the linkers can be optimized, for example, by phage-display methodsto improve the antigen binding and production yield of theantigen-binding polypeptide dimer. Examples of peptide linkers suitablefor a tandem diabody in some embodiments are GGSGGS (SEQ ID NO:95), GGSG(SEQ ID NO:96), or GGSGG (SEQ ID NO:97).

Non-limiting examples of tandem diabodies as described herein are tandemdiabodies having an anti-CD33 VL and VH domain, an anti-CD3 VL and VHdomain, domain order and linker according to Table 7.

TABLE 7 Exemplary CD33/CD3 Tandem Diabodies (TandAbs) TandemAnti-CD33 domain Anti-CD3 domain Domain Linker Diabody VL VH VH VL OrderL1/L3 L2 01 SEQ ID SEQ ID SEQ ID SEQ ID 1 GGSGGS GGSG NO: 2 NO: 12NO: 65 NO: 69 02 SEQ ID SEQ ID SEQ ID SEQ ID 1 GGSGGS GGSG NO: 3 NO: 13NO: 65 NO: 69 03 SEQ ID SEQ ID SEQ ID SEQ ID 1 GGSGGS GGSG NO: 4 NO: 14NO: 65 NO: 69 04 SEQ ID SEQ ID SEQ ID SEQ ID 1 GGSGGS GGSG NO: 5 NO: 15NO: 65 NO: 69 05 SEQ ID SEQ ID SEQ ID SEQ ID 1 GGSGGS GGSG NO: 4 NO: 14NO: 65 NO: 69 G 06 SEQ ID SEQ ID SEQ ID SEQ ID 1 GGSGGS GGSG NO: 5NO: 15 NO: 65 NO: 69 G 07 SEQ ID SEQ ID SEQ ID SEQ ID 1 GGSGGS GGSGNO: 1 NO: 11 NO: 64 NO: 68 GS 08 SEQ ID SEQ ID SEQ ID SEQ ID 3 GGSGGSGGSG NO: 2 NO: 12 NO: 64 NO: 68 GS 09 SEQ ID SEQ ID SEQ ID SEQ ID 1GGSGGS GGSG NO: 2 NO: 12 NO: 66 NO: 70 10 SEQ ID SEQ ID SEQ ID SEQ ID 1GGSGGS GGSG NO: 4 NO: 14 NO: 66 NO: 70 11 SEQ ID SEQ ID SEQ ID SEQ ID 1GGSGGS GGSG NO: 5 NO: 15 NO: 66 NO: 70 12 SEQ ID SEQ ID SEQ ID SEQ ID 1GGSGGS GGSG NO: 3 NO: 13 NO: 64 NO: 68 13 SEQ ID SEQ ID SEQ ID SEQ ID 1GGSGGS GGSG NO: 3 NO: 13 NO: 67 NO: 71 14 SEQ ID SEQ ID SEQ ID SEQ ID 1GGSGGS GGSG NO: 2 NO: 12 NO: 64 NO: 68 15 SEQ ID SEQ ID SEQ ID SEQ ID 1GGSGGS GGSG NO: 4 NO: 14 NO: 64 NO: 68 16 SEQ ID SEQ ID SEQ ID SEQ ID 1GGSGGS GGSG NO: 5 NO: 15 NO: 64 NO: 68 17 SEQ ID SEQ ID SEQ ID SEQ ID 1GGSGGS GGSG NO: 7 NO: 17 NO: 64 NO: 68 18 SEQ ID SEQ ID SEQ ID SEQ ID 2GGSGGS GGSG NO: 7 NO: 17 NO: 64 NO: 68 19 SEQ ID SEQ ID SEQ ID SEQ ID 1GGSGGS GGSG NO: 6 NO: 16 NO: 64 NO: 68 20 SEQ ID SEQ ID SEQ ID SEQ ID 1GGSGGS GGSG NO: 6 NO: 16 NO: 67 NO: 71 21 SEQ ID SEQ ID SEQ ID SEQ ID 1GGSGGS GGSG NO: 8 NO: 18 NO: 64 NO: 68 22 SEQ ID SEQ ID SEQ ID SEQ ID 1GGSGGS GGSG NO: 9 NO: 19 NO: 64 NO: 68 23 SEQ ID SEQ ID SEQ ID SEQ ID 1GGSGGS GGSG NO: 9 NO: 19 NO: 67 NO: 71 24 SEQ ID SEQ ID SEQ ID SEQ ID 1GGSGGS GGSG NO: 10 NO: 20 NO: 64 NO: 68

In some embodiments, a tandem diabody is tandem diabody 01 (SEQ IDNO:98), 02 (SEQ ID NO:99), 03 (SEQ ID NO: 100), 04 (SEQ ID NO:101), 05(SEQ ID NO: 102), 06 (SEQ ID NO: 103), 07 (SEQ ID NO: 104), 08 (SEQ IDNO: 105), 09 (SEQ ID NO: 106), 10 (SEQ ID NO: 107), 11 (SEQ ID NO:108),12 (SEQ ID NO:109), 13 (SEQ ID NO:110), 14 (SEQ ID NO:111), 15 (SEQ IDNO: 112), 16 (SEQ ID NO: 113), 17 (SEQ ID NO: 114), 18 (SEQ ID NO: 115),19 (SEQ ID NO: 116), 20 (SEQ ID NO: 117), 21 (SEQ ID NO:118), 22 (SEQ IDNO: 119), 23 (SEQ ID NO:120), or 24 (SEQ ID NO: 121) as depicted in FIG.9B to 9Y.

The CD33 binding protein and the CD33/CD3 bispecific binding protein(e.g., CD33/CD3 bispecific tandem diabody) described herein is produced,in some embodiments, by expressing polynucleotides encoding thepolypeptide of the tandem diabody which associates with anotheridentical polypeptide to form the antigen-binding tandem diabody.Therefore, another aspect is a polynucleotide, e.g. DNA or RNA, encodingthe polypeptide of an antigen-binding tandem diabody as describedherein.

The polynucleotide is constructed by known methods such as by combiningthe genes encoding at least four antibody variable domains eitherseparated by peptide linkers or, in other embodiments, directly linkedby a peptide bond, into a single genetic construct operably linked to asuitable promoter, and optionally a suitable transcription terminator,and expressing it in bacteria or other appropriate expression systemsuch as, for example CHO cells. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Thepromoter is selected such that it drives the expression of thepolynucleotide in the respective host cell.

In some embodiments, the polynucleotide is inserted into a vector,preferably an expression vector, which represents a further embodiment.This recombinant vector can be constructed according to known methods.

A variety of expression vector/host systems may be utilized to containand express the polynucleotide encoding the polypeptide of the describedantigen-binding tandem diabody. Examples of expression vectors forexpression in E. coli are pSKK (Le Gall et al., J Immunol Methods.(2004) 285(1): 111-27) or pcDNA5 (Invitrogen) for expression inmammalian cells.

Thus, the antigen-binding tandem diabody as described herein, in someembodiments, is produced by introducing a vector encoding thepolypeptide as described above into a host cell and culturing said hostcell under conditions whereby the polypeptide chains are expressed, maybe isolated and, optionally, further purified.

In other aspects, the CD33 binding protein or the CD33/CD3 bispecificbinding protein (e.g., CD33/CD3 bispecific tandem diabody) describedherein has a modification. Typical modifications include, but are notlimited to, acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphatidylinositol, drug conjugation, cross-linking, cyclization,disulfide bond formation, demethylation, formation of covalentcrosslinks, formation of cystine, formation of pyroglutamate,formylation, gamma carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. In furtherembodiments, the CD33 binding protein or the CD33/CD3 bispecific bindingprotein is modified with additional amino acids, such as a leader orsecretory sequence or a sequence for purification of the polypeptide.

In other aspects, provided herein are pharmaceutical compositionscomprising the CD33 binding protein, an antigen-binding tandem diabody,a vector comprising the polynucleotide encoding the polypeptide of theantigen binding tandem diabody or a host cell transformed by this vectorand at least one pharmaceutically acceptable carrier. The term“pharmaceutically acceptable carrier” includes, but is not limited to,any carrier that does not interfere with the effectiveness of thebiological activity of the ingredients and that is not toxic to thepatient to whom it is administered. Examples of suitable pharmaceuticalcarriers are well known in the art and include phosphate buffered salinesolutions, water, emulsions, such as oil/water emulsions, various typesof wetting agents, sterile solutions etc. Such carriers can beformulated by conventional methods and can be administered to thesubject at a suitable dose. Preferably, the compositions are sterile.These compositions may also contain adjuvants such as preservative,emulsifying agents and dispersing agents. Prevention of the action ofmicroorganisms may be ensured by the inclusion of various antibacterialand antifungal agents.

Bispecific CD33/CD3 binding proteins with high-affinity binding to CD33and CD3 are highly active in a large number of primary AML specimens,suggesting that these molecules could be active against human AML acrossthe entire cytogenetic/molecular disease spectrum, even in cases ofminimal CD33 expression. Of note, drug-specific cytotoxicity is alsoobserved in the presence of residual autologous T-cells and issignificantly augmented by the addition of controlled amounts of healthydonor T-cells (see Example 6).

The CD33/CD3 bispecific binding proteins, in particular tandemdiabodies, can induce potent cytolysis of CD33⁺ leukemic cells in vitro.The data indicate that high-affinity binding to both CD33 and CD3maximizes bispecific protein-induced T-cell activation and anti-AMLefficacy. High-affinity CD33/CD3-directed bispecific binding proteins,such as the tandem diabodies described herein display cytolytic activityin primary AML in vitro. Thus, these bispecific binding proteins andtandem diabodies are suitable for a therapeutic approach for thetreatment of acute myeloid leukemia (AML) or other hematologicmalignancies, for example, myeloid dysplastic syndrome (MDS) ormyeloproliferative disease (MPD).

Therefore, provided herein are methods wherein the antigen-bindingtandem diabody as described herein above is administered in an effectivedose to a subject, e.g., a patient, for the treatment of a CD33⁺ cancer(e.g. acute myeloid leukemia (AML)), disease or condition. CD33⁺ cancersinclude, but are not limited to, acute leukemias such as acute myeloidleukemia, acute lymphoblastic leukemia (ALL) including precursor B-celllymphoblastic leukemia, myeloid sarcoma, multiple myeloma, acutelymphomas such as acute lymphoblastic lymphoma, chronic myelomonocyticleukemia and the like. CD33⁺ diseases and conditions include immunesuppressive states or environments attributed by myeloid derivedsuppressor cells (MDSCs) in certain cancers and chronic inflammation.

In some embodiments, the antigen-binding tandem diabody as describedherein is administered for the treatment of acute myeloid leukemia(AML). In certain embodiments, the antigen-binding tandem diabody asdescribed herein is administered for the treatment of an acute myeloidleukemia subtype.

The French-American-British classification system divides AML into eightsubtypes: AML-M0 (minimally differentiated), AML-M1 (withoutmaturation), AML-M2 (with granulocytic maturation), AML-M3(promyelocytic or acute promyelocytic leukemia), AML-4 (acutemyelomonocytic leukemia), AML-M5 (acute monoblastic or monocyticleukemia), AML-M6 (acute erythroid leukemia), and AML-M7 (acutemegakaryoblastic leukemia). In certain instances, the antigen-bindingtandem diabody as described herein is administered for the treatment ofAML-M0, AML-M1, AML-M2, AML-M3, AML-M4, AML-M5, AML-M6, or AML-M7.

The WHO AML classification scheme organizes AML according to thefollowing subtypes: AML with Recurrent Genetic Abnormalities, AML withmyelodysplasia-related changes, Therapy-related myeloid neoplasms,Myeloid sarcoma, Myeloid proliferations related to Down syndrome,Blastic plasmacytoid dendritic cell neoplasm, and AML not otherwisecategorized. In certain other instances, the antigen-binding tandemdiabody as described herein is administered for the treatment of AMLwith Recurrent Genetic Abnormalities, AML with myelodysplasia-relatedchanges, Therapy-related myeloid neoplasms, Myeloid sarcoma, Myeloidproliferations related to Down syndrome, Blastic plasmacytoid dendriticcell neoplasm, or AML not otherwise categorized.

In some other embodiments, the antigen-binding tandem diabody asdescribed herein is administered for the treatment of a newly diagnosed,recurrent or refractory AML.

In further embodiments, the antigen-binding tandem diabody as describedherein is administered for the treatment of a preleukemia blood disordersuch as myeloid dysplastic syndrome (MDS) or myeloproliferative disease(MPD). In certain instances, the antigen-binding tandem diabody asdescribed herein is administered for the treatment of MDS. In certaininstances, the antigen-binding tandem diabody as described herein isadministered for the treatment of MPD.

In other embodiments, the antigen-binding tandem diabody as describedherein is administered for the treatment of multiple myeloma. In furtherembodiments, the antigen-binding tandem diabody as described herein isadministered for the treatment of chronic myelomonocytic leukemia(CMML).

In other embodiments, the antigen-binding tandem diabody as describedherein is administered for inhibiting or eliminating myeloid derivedsuppressor cells (MDSCs). MDSCs highly overexpress CD33 in certainisolated diseased tissues and possess strong immunosuppressiveactivities. In certain human cancers (CD33⁺ as well as non-CD33+), MDSCsproliferate and are activated to suppress tumor-specific CD4⁺ T-cellresponses and induce T_(reg) cells, allowing the tumor or cancer toflourish in a microenvironment. In chronic inflammation, MDSCs arereportedly expanded and found at inflammation sites to suppress T cellimmune function. In other embodiments, the antigen-binding tandemdiabody as described herein is administered for treating a conditionassociated with MDSCs. In yet other embodiments, the antigen-bindingtandem diabody as described herein is administered to treat immunesuppression. In yet other embodiments, the antigen-binding tandemdiabody as described herein is administered to treat inflammationsuppressed by MDSCs. In yet other embodiments, the antigen-bindingtandem diabody as described herein is administered to treat a decreasedimmune response caused by MDSCs. In yet other embodiments, theantigen-binding tandem diabody as described herein is administered totreat angiogenesis, tumor invasion, or metastasis of cancers that arepromoted by MDSCs. In yet other embodiments, the antigen-binding tandemdiabody as described herein is administered to treat a cancer or tumorthat is enhanced, augmented, aggravated or increased by MDSCs.

The antigen-binding tandem diabody described herein is contemplated foruse as a medicament. Administration is effected by different ways, e.g.by intravenous, intraperitoneal, subcutaneous, intramuscular, topical orintradermal administration. In some embodiments, the route ofadministration depends on the kind of therapy and the kind of compoundcontained in the pharmaceutical composition. The dosage regimen will bedetermined by the attending physician and other clinical factors.Dosages for any one patient depends on many factors, including thepatient's size, body surface area, age, sex, the particular compound tobe administered, time and route of administration, the kind of therapy,general health and other drugs being administered concurrently. An“effective dose” refers to amounts of the active ingredient that aresufficient to affect the course and the severity of the disease, leadingto the reduction or remission of such pathology. An “effective dose”useful for treating and/or preventing AML may be determined using knownmethods.

In further embodiments, the antigen-binding tandem diabody describedherein is administered in combination with a standard therapy to CD33⁺cancers, diseases or conditions. Standard therapies includechemotherapies, immunotherapies, hormone therapies, radiation, surgery,gene therapies and the like. In certain instances, the antigen-bindingtandem diabody described herein is administered in combination with astandard AML therapy. In certain instances, the antigen-binding tandemdiabody described herein is administered in combination with cytarabine,azacitidine, decitabine, an anthracycline (e.g., daunorubicin,idarubicin, doxorubicin, and the like), amsacrine, fludarabine,clofarabine, cladribine, nelarabine, methotrexate, bortezomib,carfilzomib, melphalan, ibrutinib, thalidomide, lenalidomide,pomalidomide, apremilast, an epipodophyllotoxin (e.g., etoposide,teniposide, and the like), an anthracenedione (e.g., mitoxantrone,pixantrone, losoxantrone, piroxantrone, ametantrone and the like) ananti-CD20 agent (e.g., rituximab, ocrelizumab, ofatumumab, orcombinations thereof. In certain instances, the antigen-binding tandemdiabody described herein is administered in combination with cytarabine(ara-C). In certain instances, the antigen-binding tandem diabodydescribed herein is administered in combination with azacitidine. Incertain instances, the antigen-binding tandem diabody described hereinis administered in combination with decitabine. In further instances,the antigen-binding tandem diabody described herein is administered incombination with an anthracycline (e.g., daunorubicin, idarubicin,doxorubicin, and the like). In other instances, the antigen-bindingtandem diabody described herein is administered in combination with acheckpoint inhibitor (e.g., PD-1 inhibitor, CTLA-4 inhibitor, and thelike). In yet other instances, the antigen-binding tandem diabodydescribed herein is administered in combination with anepipodophyllotoxin (e.g., etoposide, teniposide, and the like). In yetother instances, the antigen-binding tandem diabody described herein isadministered in combination with an anthracenedione (e.g., mitoxantrone,pixantrone, losoxantrone, piroxantrone, ametantrone and the like).

The examples below further illustrate the described embodiments withoutlimiting the scope of the invention.

Example 1 Cloning of DNA Expression Constructs Encoding Single-Chain FvAntibodies

For bacterial expression of anti-CD33 single-chain Fv (scFv) antibodiesin E. coli, DNA coding sequences of all molecules were cloned into abacterial expression vector. All expression constructs were designed tocontain coding sequences for an N-terminal signal peptide and C-terminalhexa-histidine (6×His)-tag to facilitate antibody secretion into theperiplasm and purification, respectively. The amino acid sequences ofthe VL and VH-domains from all anti-CD33 scFv clones are shown in Table3 and Table 4.

Expression of Recombinant Anti-CD33 scFv Antibodies in E. coli

Recombinant scFv antibodies were expressed as soluble secreted proteinsin the E. coli periplasm. In a first step a small medium culturesupplemented with ampicillin was inoculated with transformed bacteriaand incubated for 16 h at 28° C. Subsequently, optical density wasadjusted by adding a second medium supplemented with ampicillin andincubated once more at 28° C. until an optical density in the range of0.6-0.8 at 600 nm was reached. Protein expression was induced throughaddition of 50 μM IPTG and incubation of cultures at 21-28° C. and 200rpm for up to 16 h. Following incubation, cells were pelleted (30 min,4° C., 7500 rpm) and stored at −20° C. until further processing.

Purification of Anti-CD33 Single-Chain Fv Antibodies

Recombinant scFv were extracted from E. coli periplasm followingcentrifugation of bacterial cell cultures by resuspending cell pelletsin buffer and incubation for 30 min at room temperature with gentleagitation. Cells were pelleted and supernatants containing recombinantproteins were kept. The procedure was repeated once more beforesupernatants were pooled and homogenized by ultrasonication. Homogenateswere diluted, supplemented with low concentrations of imidazole andloaded onto a prepacked immobilized metal affinity chromatography (IMAC)column (GE Healthcare). The column was washed until baseline was reachedand bound protein was then eluted with an imidazole buffer. Antibodycontaining fractions were pooled and subsequently purified bysize-exclusion chromatography (SEC). Finally, protein eluates wereconcentrated by ultrafiltration and dialysed against storage buffer.Subsequent to low pH treatment (incubation at pH 3.0 for 20-24 h at 37°C.), samples were neutralized using Tris. Purified proteins were storedas aliquots at −80° C. until use.

Example 2 Cloning of DNA Expression Constructs Encoding Tandem Diabodies(TandAb®)

For expression of bispecific tandem diabodies in CHO cells, codingsequences of all molecules were cloned into a mammalian expressionvector system. The anti-CD33 scFv domains of Example 1 were used toconstruct CD33/CD3 tandem diabodies in combination with an anti-CD3 scFvdomain, with domains organized as shown in Table 7 and FIG. 3. In brief,gene sequences encoding anti-CD33 VH and VL domains separated by apeptide linker (VH-linker-VL or VL-linker-VH) were synthesized andsubcloned. The resulting construct was digested to generate separate VHand VL coding sequences utilizing a Bam HI restriction site locatedwithin the linker sequence. These VH and VL fragments were then ligatedwith a DNA fragment encoding VH and VL domains of anti-CD3 (VH-linker-VLor VL-linker-VH) to yield the final construct. Domain order variants 1to 3 of CD33/CD3 tandem diabodies are shown in FIG. 3. All expressionconstructs were designed to contain coding sequences for an N-terminalsignal peptide and a C-terminal hexahistidine (6×His)-tag to facilitateantibody secretion and purification, respectively.

Expression of Tandem Diabodies in Stably Transfected CHO Cells

A CHO cell expression system (Flp-In®, Life Technologies), a derivativeof CHO-K1 Chinese Hamster ovary cells (ATCC, CCL-61) (Kao and Puck,Proc. Natl. Acad Sci USA 1968; 60(4):1275-81), was used. Adherent cellswere subcultured according to standard cell culture protocols providedby Life Technologies.

For adaption to growth in suspension, cells were detached from tissueculture flasks and placed in serum-free medium. Suspension-adapted cellswere cryopreserved in medium with 10% DMSO.

Recombinant CHO cell lines stably expressing secreted tandem diabodieswere generated by transfection of suspension-adapted cells. Duringselection with the antibiotic Hygromycin B viable cell densities weremeasured twice a week, and cells were centrifuged and resuspended infresh selection medium at a maximal density of 0.1×10⁶ viable cells/mL.Cell pools stably expressing tandem diabodies were recovered after 2-3weeks of selection at which point cells were transferred to standardculture medium in shake flasks. Expression of recombinant secretedproteins was confirmed by performing protein gel electrophoresis or flowcytometry. Stable cell pools were cryopreserved in DMSO containingmedium.

Tandem diabodies were produced in 10-day fed-batch cultures of stablytransfected CHO cell lines by secretion into the cell culturesupernatant. Cell culture supernatants were harvested after 10 days atculture viabilities of typically >75%. Samples were collected from theproduction cultures every other day and cell density and viability wereassessed. On day of harvest, cell culture supernatants were cleared bycentrifugation and vacuum filtration before further use.

Protein expression titers and product integrity in cell culturesupernatants were analyzed by SDS-PAGE.

Purification of Tandem Diabodies

Tandem diabodies were purified from CHO cell culture supernatants in atwo-step procedure. The His6-tagged constructs were subjected to Ni-NTASuperflow chromatography in a first step followed by preparative sizeexclusion chromatography (SEC) on Superdex 200 in a second step. Elutedtandem diabodies were characterized with regards to their homodimer(tandem diabody) content and pooled if the homodimer content was 90% orhigher. Finally, pooled samples were buffer-exchanged and concentratedby ultrafiltration to a typical concentration of >1 mg/mL. Purity andhomogeneity (typically >90%) of final samples were assessed by SDS PAGEunder reducing and non-reducing conditions, followed by immunoblottingusing an anti-His-Tag antibody as well as by analytical SEC,respectively. Purified proteins were stored at aliquots at −80° C. untiluse.

Polypeptides of CD33/CD3 tandem diabodies are shown in Table 7 and FIG.3. Each tandem diabody consists of two identical polypeptides (FIG. 1).Both outer linkers L1 and L3 were comprised of six amino acids GGSGGS(SEQ ID NO:95), whereas the central peptide linker 2 varied in length(4-6 amino acids) with the sequences GGSG (SEQ ID NO:96), GGSGG (SEQ IDNO:97), or GGSGGS (SEQ ID NO:95), respectively.

Using a series of anti-CD33 variable domains and anti-CD3 variabledomains a large number of tandem diabody molecules was generated thatcould be stably produced in transfected cell lines and that maintainedstability at body temperature as well as after repeated freeze/thawcycles. To facilitate further development and preclinical toxicologystudies, emphasis was placed on the selection of tandem diabodymolecules that showed binding to both human and cynomolgus monkey CD33.Examples of complete amino acid sequences are shown for the single-chainof tandem diabodies 12 (SEQ ID NO: 109), 14 (SEQ ID NO: 111) and 16 (SEQID NO: 113) in FIGS. 9M, 9O and 9Q, respectively. In this example theorder of the variable domains and their linkers for the structures is:VL (CD3)-L1-VH (CD33)-L2-VL (CD33)-L3-VH (CD3).

Example 3 Determination of Antibody Affinity by Flow Cytometry

Cells were incubated with 100 μL of serial dilutions of CD33/CD3 tandemdiabodies. After washing three times with FACS buffer the cells wereincubated with 0.1 mL of 10 μg/mL mouse monoclonal anti-His antibody inthe same buffer for 45 min on ice. After a second washing cycle, thecells were incubated with 0.1 mL of 15 μg/mL FITC-conjugated goatanti-mouse IgG antibodies under the same conditions as before. As acontrol, cells were incubated with the anti-His IgG followed by theFITC-conjugated goat anti-mouse IgG antibodies without anti-CD33 tandemdiabodies. The cells were then washed again and resuspended in 0.2 mL ofFACS buffer containing 2 μg/mL propidium iodide (PI) in order to excludedead cells. The fluorescence of 1×10⁴ living cells was measured using aBeckman-Coulter FC500 MPL flow cytometer using the MXP software(Beckman-Coulter, Krefeld, Germany) or a Millipore Guava EasyCyte flowcytometer using the Incyte software (Merck Millipore, Schwalbach,Germany). Mean fluorescence intensities of the cell samples werecalculated using CXP software (Beckman-Coulter, Krefeld, Germany) orIncyte software (Merck Millipore, Schwalbach, Germany). Aftersubtracting the fluorescence intensity values of the cells stained withthe secondary and tertiary reagents alone the values were used forcalculation of the K_(D) values with the equation for one-site binding(hyperbola) of the GraphPad Prism (version 6.00 for Windows, GraphPadSoftware, La Jolla Calif. USA).

The tandem diabodies were tested for their binding affinities to humanCD3⁺ and CD33⁺ cells and cynomolgus CD3⁺ and CD33⁺ cells. Exemplarybinding data for selected tandem diabodies are summarized in Table 8:

TABLE 8 CD3 and CD33 binding characteristics of CD33/CD3 tandemdiabodies: EC₅₀ on K_(D) on T K_(D) on HL- K_(D) on KG- K_(D) on U-937K_(D) ratio HL-60 TandAb cells [nM] 60 [nM] 1 [nM] [nM] cynoCD33/huCD33[pM] 01 94.2 0.6 0.9 7.1 0.7 1.9 02 69.8 0.2 0.3 0.9 1.1 0.5 03 81.9 1.11.8 8.9 0.6 3.6 04 79.3 0.5 0.5 1.7 1.1 1.8 05 69.5 1.0 1.2 6.2 0.8 2.706 86.3 0.4 0.5 1.6 0.8 1.6 07 49.7 13.7 47.9 47.1 45.8 17.8 08 2.4 0.30.5 1.8 0.6 1.8 09 2.4 0.5 0.3 2.2 1.0 6.8 10 1.9 0.5 1.0 1.7 0.8 7.0 112.6 0.3 0.5 0.6 1.2 5.9 12 1.5 0.3 0.9 0.5 1.7 1.3 13 55.7 0.2 0.3 0.51.6 1.1 14 2.1 0.3 0.3 1.2 1.0 1.6 15 1.3 0.4 0.3 0.9 1.1 1.8 16 2.1 0.30.2 0.3 1.4 1.5 17 3.3 5.0 52.5 24.4 1.9 18.4 18 1.9 3.4 16.3 15.1 3.16.3 19 6.3 2.8 3.6 5.4 37.3 5.7 20 143.8 4.1 7.0 7.2 33.8 10.0 21 2.19.7 25.1 80.2 0.9 7.6 22 4.1 0.7 2.0 8.6 0.6 3.2 23 97.2 0.4 1.0 5.1 1.92.8 24 2.3 5.6 12.4 39.5 1.8 9.6

#K_(D) ratio cyno CD33/human CD33 was calculated based on the K_(D)values measured on CHO cells expressing cynomolgus CD33 and human CD33,respectively. _(‡)K_(D) ratio hu CD3/hu CD33 was calculated based on theK_(D) values measured on Jurkat cells (hu CD3) and the mean K_(D) ofthree human CD33⁺ tumor cell lines (HL-60, KG-1, U937).

CD3 binding affinity and crossreactivity were evaluated in titration andflow cytometric experiments on CD3⁺ Jurkat cells (provided by Dr.Moldenhauer, DKFZ Heidelberg; human acute T-cell leukemia) and thecynomolgus CD3⁺ HSC-F cell line (JCRB, cat.: JCRB 1164). CD33 bindingand crossreactivity were assessed on the human CD33⁺ tumor cell lines:HL-60 (DSMZ, cat.: ACC 3, human B cell precursor leukemia), U-937 (DSMZ,cat.: ACC5; human histiocytic lymphoma), and KG-1 (DSMZ, cat.: ACC14;acute myeloid leukemia). The K_(D) ratio of crossreactivity wascalculated using the K_(D) values determined on the CHO cell linesexpressing either recombinant human or recombinant cynomolgus antigens.

The tandem diabodies exhibited a relatively high affinity to human CD33⁺on most of the tested tumor cell lines below 1 nM. Affinities to humanCD3 were determined to be equal or less than 2 nM.

Example 4 Cytotoxicity Assay

For the cytoxicity assay target cells cultured under standard conditionswere harvested, washed and resuspended in diluent C, provided in thePKH67 Green Fluorescent Cell Linker Mini Kit, to a density of 2×10⁷cells/mL. The cell suspension was then mixed with an equal volume of adouble concentrated PKH67-labeling solution and incubated for 2-5 min atRT. The staining reaction was performed by adding an equal volume of FCSand incubating for 1 min. After washing the labeled target cells withcomplete RPMI medium, cells were counted and resuspended to a density of2×10⁵ cells/mL in complete RPMI medium. 2×10⁴ target cells were thenseeded together with enriched human T-cells as effector cells at an E:Tratio of 5:1, in the presence of increasing concentrations of theindicated tandem diabodies in individual wells of a microtiter plate, ina total volume of 200 μL/well. Spontaneous cell death and killing oftargets by T-cells in the absence of antibodies were determined for atleast three replicates on each plate. After centrifugation the assayplates were incubated for the indicated periods of time at 37° C. in ahumidified atmosphere with 5% CO₂. After incubation, cultures werewashed once with FACS buffer and then resuspended in 150 L FACS buffersupplemented with 2 μg/mL PI. The absolute amount of living target cellswas measured by a positive green staining with PKH67 and negativestaining for PI using a Beckman-Coulter FC500 MPL flow cytometer(Beckman-Coulter) or a Millipore Guava EasyCyte flow cytometer (MerckMillipore). Based on the measured remaining living target cells, thepercentage of specific cell lysis was calculated according to thefollowing formula: [1-(number of living targets_((sample))/number ofliving targets_((spontaneous)))]×100%. Sigmoidal dose response curvesand EC₅₀ values were calculated by non-linear regression/4-parameterlogistic fit using the GraphPad Software. The lysis values obtained fora given antibody concentration were used to calculate sigmoidaldose-response curves by 4 parameter logistic fit analysis using thePrism software.

EC₅₀ values were determined in 20-24 hour assay on CD33⁺ U-937 (DSMZ,cat.: ACC5; human histiocytic lymphoma) target cells with enriched humanT-cells as effector cells at a ratio of 5:1. Some tandem diabodies werealso tested in cytotoxicity assays on CD33⁺ KG-1 (DSMZ, cat.: ACC14;acute myeloid leukemia) and HL-60 target cells. Specifically, HL-60cells were chosen as a model of an AML with relatively high cell surfaceexpression of CD33 (arbitrary MFI [mean±SEM]: 3,133±215; n=3), and KG-1awas chosen as a model of an AML with very limited CD33 expression(arbitrary MFI: 277±11; n=3). Exemplary cytotoxicity data for selectedtandem diabodies are summarized in Table 9. Additional cytotoxicity datafor HL-60 cell lines is found on Table 8, last column.

TABLE 9 In vitro potency of CD33/CD3 tandem diabodies on different CD33⁺tumor cell lines: EC₅₀ [pM (pg/mL)] on human Tandem CD33⁺ target celllines diabody HL-60 U-937 KG-1 mean 12 1.3 (137) 0.8 (84)  1.2 (126) 1.1(116) 14 1.6 (168) 3.6 (378) 2.6 (273) 2.6 (273) 16 1.5 (158) 1.9 (200)1.8 (189) 1.7 (179)

EC₅₀ values were determined in FACS-based cytotoxicity assays withprimary human T-cells as effector cells at an E:T ratio of 5:1 on theindicated target cell lines incubated for 20-24 hours Each tandemdiabody was tested on each tumor cell line in at least two independentexperiments. Mean values are presented.

Example 5 Further Cytotoxicity Screening Experiments in Human CD33+ AMLCell Lines at 48 Hours

As described above significant cytotoxicity was detected as early as 24hours, however higher levels of toxicity can be detected at 48 hours.For the subsequent assays a 48-hour time point was chosen. The impact ofT-cell selection on tandem diabody-induced cytotoxicity was tested. Toaccomplish this, unstimulated PBMCs from a healthy volunteer donor wereobtained, and CD3⁺ cells were isolated both by simple “positiveenrichment” via use of CD3 microbeads as well as by more complex“negative selection” via a microbead cocktail of antibodies againstCD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a. As depictedin FIG. 4, tandem diabody-induced cytotoxicity was greater withnegatively selected healthy donor T-cells than positively selectedT-cells. However, the relative cytotoxic activities of individual tandemdiabodies were unaffected by the method of T-cell selection. Thereforethe subsequent assays were performed with positively enriched healthydonor T-cells.

Unstimulated mononuclear cells were collected from healthy adultvolunteers via leukapheresis by the Fred Hutchinson Cancer ResearchCenter (FHCRC) Hematopoietic Cell Processing Core (Core Center ofExcellence) under research protocols approved by the FHCRC InstitutionalReview Board. T-cells were enriched through magnetic cell sorting eithervia CD3 Microbeads (“positive enrichment”) or via Pan T-Cell IsolationKit (“negative selection”; both from Miltenyi Biotec, Auburn, Calif.),and then frozen in aliquots and stored in liquid nitrogen. Thawed cellaliquots were labeled with 3 μM CellVue Burgundy (eBioscience, SanDiego, Calif.) according to the manufacturer's instructions. PurifiedPBMCs were cultured in the presence of various concentrations of tandemdiabody molecules.

For the quantification of drug-induced cytotoxicity cells were incubatedat 37° C. (in 5% CO₂ and air), as in Example 4, at different E:T cellratios. After 24-72 hours, cell numbers and drug-induced cytotoxicity,using DAPI to detect non-viable cells, were determined using a LSRIIcytometer (BD Biosciences) and analyzed with FlowJo. AML cells wereidentified by forward/side scatter properties and, in experiments wherehealthy donor T-cells were added, negativity for CellVue Burgundy dye(FIG. 5). Drug-induced specific cytotoxicity is presented as: %cytotoxicity=100× (1−live target cells_(treated)/live targetcells_(control)). Results from cytotoxicity assays are presented as meanvalues±standard error of the mean (SEM). Spearman nonparametriccorrelation was used to compute correlations between continuous samplecharacteristics. All P-values are two-sided. Statistical analyses wereperformed using GraphPad Prism software.

In the absence of healthy donor T-cells, neither of the CD33/CD tandemdiabodies exerted any noticeable cytotoxic effect on AML cell lines inthe absence of T-cells, confirming the absolute requirement for T-cellsfor their cytotoxic effects (data not shown). In the presence ofT-cells, the extent of tandem diabody-induced specific cytotoxicity wasdependent on the concentration of the tandem diabody as well as the E:Tcell ratio. Direct head-to-head comparisons between theCD33/CD3-directed tandem diabody molecules and one control tandemdiabody (00) indicated considerable differences in antibody-inducedcytotoxicity in both HL-60 cells (FIG. 6A/B and Table 10) and KG-1acells (FIG. 6C/D and Table 10), with results being highly reproduciblein repeat experiments. Overall, the degree of tandem diabody-inducedcytotoxicity correlated with the binding affinity for CD3 on primaryhuman T-cells (for cytotoxicity in KG-1a cells at 25 pM (approx. 2.5ng/mL) and E:T=5:1: r=−0.542, p=0.009; for cytotoxicity in HL-60 cellsat 25 pM and E:T=5:1: r=−0.391, p=0.07). The tandem diabodies 12, 14, 16were highly cytotoxic for both HL-60 and KG-1a cells.

TABLE 10 CD25 and CD69 induction and cytotoxicity at 48 h of CD33/CD3tandem diabodies CD3 K_(D) CD33 K_(D) T cell (nM) (nM) CD25 CD69Proliferation in Cytotoxicity Cytotoxicity Tandem Human HL-60 InductionInduction PBMC EC₅₀ HL-60 cells KG-1a cells Diabody¹ T-cells cells EC₅₀(pM)² EC₅₀ (pM)² (pM)³ (% ± SEM)⁴ (% ± SEM)⁴ 15 1.3 0.4 6 7 7 82.9 ± 3.780.2 ± 1.9  12 1.5 0.3 6 3 2 84.7 ± 2.3 85.6 ± 1.6  10 1.9 0.5 10 6 648.0 ± 2.4 78.6 ± 2.3  14 2.1 0.3 10 7 6 86.0 ± 0.4 69.8 ± 5.7  21 2.19.7 ND 225 500 12.4 ± 1.0 0.0 ± 0.2 24 2.3 5.6 ND 57 264 24.5 ± 1.9 1.1± 0.2 09 2.4 0.5 11 7 9  43.2 ± 15.8 74.6 ± 3.2  11 2.6 0.3 11 5 6 52.7± 8.1 84.7± 1.4  17 3.3 5.0 30 114 30  4.2 ± 0.2 0.7 ± 0.4 22 4.1 0.7 104 7 74.2 ± 7.4 44.4 ± 5.3  16 5.1 0.3 1 2 3 86.0 ± 1.4 81.3 ± 1.5  196.3 2.8 9 5 6 79.4 ± 3.5 83.8 ± 2.9  07 49.7 13.7 134 65 50  6.3 ± 3.32.1 ± 0.7 13 55.7 0.2 30 22 23 70.4 ± 2.5 1.3 ± 0.4 05 69.5 1 116 74 7423.8 ± 6.9 0.3 ± 0.3 02 69.8 0.2 42 27 4 80.9 ± 3.6 4.6 ± 2.1 04 79.30.5 94 62 44 24.1 ± 4.0 0.7 ± 0.8 03 81.9 1.1 117 87 63 13.1 ± 3.6 0.0 ±0.5 06 86.3 0.4 39 21 48 45.7 ± 6.4 1.4 ± 0.2 01 94.2 0.6 92 91 89  8.0± 1.6 0.4 ± 0.4 23 97.2 0.4 41 17 37 73.7 ± 2.6 1.5 ± 0.3 20 143.8 4.198 75 38 31.2 ± 3.9 1.1 ± 0.3 ¹Tandem Diabodies (TandAbs) are listed inorder of increasing CD3 affinity ²CD25 and CD69 induction was measuredafter 24 hours in unfractionated PBMC cultures. ³T cell proliferationinduced by CD33/CD3 tandem diabodies in unfractionated PBMC with CD33+cells present. ⁴Cytotoxicity (%) after 48 hours of DAPI+ cells at atandem diabodies concentration of 25 pM in the presence of healthy donorT-cells at an E:T cell ratio of 5:1 from 3 independent experimentsperformed in duplicate wells. ND: no CD25 activation detectable

Example 6 Further Characterization of Tandem Diabodies in Primary HumanAML Specimens

For a comprehensive characterization of the cytotoxic properties ofthese candidates, specimens from AML patients were obtained for thestudies from a FHCRC specimen repository.

Frozen aliquots of Ficoll-isolated mononuclear cells from pretreatment(“diagnostic”) peripheral blood or bone marrow specimens from adultpatients with AML were obtained from repositories at FHCRC. We used the2008 WHO criteria to define AML (Vardiman et al.; Blood. 2009;114(5):937-951) and the refined United Kingdom Medical Research Council(MRC) criteria to assign cytogenetic risk (Grimwalde et al.; Blood.2010; 116(3):354-365). Patients provided written informed consent forthe collection and use of their biospecimens for research purposes underprotocols approved by the FHCRC Institutional Review Board. Clinicaldata were de-identified in compliance with Health Insurance Portabilityand Accountability Act regulations. After thawing, cells were stainedwith directly labeled antibodies recognizing CD33 (clone P67.6;PE-Cy7-conjugated), CD3 (clone SK7; PerCP-conjugated), CD34 (clone 8G12;APC-conjugated; all from BD Biosciences, San Jose, Calif.), and CD45(clone HI30; APC-eFluor®780-conjugated; eBioscience). To identifynonviable cells, samples were stained with 4′,6-diamidino-2-phenylindole(DAPI). At least 10,000 events were acquired on a Canto II flowcytometer (BD Biosciences), and DAPI− cells analyzed using FlowJo (TreeStar, Ashland, Oreg.).

After thawing, specimens had >58% AML blasts, as determined by flowcytometry based on CD45/side-scatter properties. Specimens had >50%viable cells immediately after thawing and >50% viable cells after 48hours in cytokine-containing liquid culture (FIG. 7). Median age of thepatients was 58.1 (range: 23.9-76.2) years; cytogenetic disease risk wasfavorable in 2, intermediate in 18, and adverse in 7. Information on themutation status of NPM1, FLT3, and CEBPA was incomplete; however, onesample was known to be CEBPA^(double-mutant), and another sample wasNPM1^(pos)/FLT3-ITD^(neg). The median percentage of myeloid blasts andCD3⁺ T-cells in the studied specimens was 86.1% (range: 58.4-97.0%) and2.0% (range: 0-11.9%), respectively, and the median sample viabilityafter 48 hours in culture was 80.1% (range: 53.6-93.6%). Fifteen of thepatients had newly diagnosed AML, whereas 12 either had relapsed (n=7)or refractory (n=5) disease at the time of specimen collection. Assummarized in Table 11, basic characteristics of the specimens frompatients with newly diagnosed AML were similar to those withrelapsed/refractory disease with regard to CD33 expression on myeloidblasts, amount of autologous T-cells, proportion of myeloid blasts, andculture viability.

The addition of tandem diabody molecules to AML specimen culturesresulted in modest, dose-dependent cytotoxicity (FIG. 8A), demonstratingthat autologous T-cells, contained in the specimens from patients withactive AML, can be engaged to lyse leukemic cells. In the presence ofhealthy donor T-cells, the cytotoxic activity of individual tandemdiabodies was strictly dependent on the drug dose and the E:T cell ratio(FIG. 8B/C). However, high activity of tandem diabodies was observedeven in some specimens with very low CD33 expression on AML blasts.Among the tandem diabody molecules, 12 appeared to be the most active,since it had the highest cytotoxicity at low concentrations (2.5 pM(approx. 250 ng/mL) and, to a less pronounced degree, also 10 pM(approx. 1 ng/mL)) at both E:T=1:3 and E:T=1:1.

The CD33/CD3 tandem diabodies have been screened in representative AMLpatient blood samples, which varied in terms of patient sex, age,disease stage (newly diagnosed, relapsed, refractory), degree of CD33expression and cytogenic risk (Table 11). Remarkably, a number ofexamined tandem diabodies (e.g., 02, 08, 09, 11, 12, 14, 16, 19, 22 and23) were highly active in nearly all patient samples across the diseasespectrum as shown in FIG. 15. Moreover, the extent and scope of activityis similar in all stages of AML, including newly-diagnosed, relapsed andrefractory patients.

TABLE 11 Characteristics of primary AML specimens All patients Newlydiagnosed Relapsed/refractory (n = 27) AML (n = 15) AML (n = 12) Medianage (range), years 58.1 (23.9-76.2) 64.0 (40.2-76.2) 44.4 (23.9-67.4)Cytogenetic/molecular risk Favorable 2 2 — Intermediate 18 10 8CEBPA^(double-mutant) 1 1 — NPM1^(pos)/FLT3-ITD^(neg) 1 — 1NPM1^(pos)/FLT3-ITD^(pos) or NPM1^(neg)/FLT3-ITD^(pos) 10 5 5 Adverse 73 4 Specimen source Bone marrow 11 4 7 Peripheral blood 16 11 5 Median %blasts (range) 86.1 (58.4-97.0) 86.1 (66.7-95.5) 86.7 (58.4-97.0) MedianCD33 expression on blasts (range) 849 (5-5,356) 849 (5-5,356) 788(7-2,242) Median % T-cells (range) 2.0 (0-11.9) 1.6 (0-11.9) 2.1(0.7-8.7) Median % viability at 48 hours (range) 80.1 (53.6-93.6) 76.0(53.6-93.6) 83.5 (63.9-93.1)

Example 7 Potency and Efficacy of CD33/CD3 Tandem Diabody 12 and TandemDiabody 16 on Different CD33⁺ Cell Lines of Various Origin ExpressingDifferent Levels of CD33

In order to assess whether potency and efficacy of CD33/CD3 tandemdiabodies depend on the CD33 density on the target cells, various humanCD33⁺ tumor cell lines and CHO cells expressing recombinant human CD33were tested for their CD33 expression levels using the QIFIKITquantification kit and anti-CD33 mAb WM53. The results in Table 12 showthat the CD33 densities on the tumor cell lines were in the rangebetween ˜1300 SABC (standardized antibody binding capacity) and ˜46000SABC. The expression on CHO-CD33 cells was ˜197000 SABC, substantiallyhigher than on the tumor cell lines. All tested CD33⁺ cell lines wereused as target cells in at least 3 independent FACS-based cytotoxicityassays with human T-cells as effector cells at an effector-to-targetratio of 5:1 in the presence of serial dilutions of CD33/CD3 tandemdiabody 12 and tandem diabody 16. In each assay EC₅₀ and tandemdiabody-mediated lysis values were calculated by non-linear regression.The results demonstrate that neither the potency (EC₅₀ values) nor theefficacy (% lysis) of 12 and 16 correlates with the CD33 density on thesurface of target cells.

Noteworthy, at least 12 and 16 exhibit their cytotoxic activity alsoagainst cells like SEM with very low CD33 densities of below 1500 SABC.

TABLE 12 CD33 target cell surface expression and cytotoxic potency ofCD33/CD3 tandem diabody 12 and tandem diabody 16: CD33 density 12 16[SABC] EC₅₀ [pM] EC₅₀ [pM] Cell line mean SD mean SD mean SD CHO-CD33196990 28053 11.8 11.2 24.0 19.5 HL-60 45948 4478 1.4 0.5 1.6 0.4 KG-142828 6923 1.0 0.6 1.9 2.0 KASUMI-1 25922 6484 1.3 0.6 2.4 1.4 THP-122065 415 1.9 0.2 6.0 1.2 RPMI-8226 19931 2604 14.0 17.8 2.8 2.0 U-93717669 4593 0.9 0.1 1.3 0.6 K562 13789 2156 4.5 1.3 4.8 2.7 BV-173 85181231 1.4 0.6 3.2 1.6 SEM 1306 144.2 2.2 0.5 5.1 3.0

The standardized antibody binding capacity (SABC) on CD33⁺ cell lineswas determined using QIFIKIT and the anti-CD33 mAb WM53. EC₅₀ values fortandem diaboody 12 and tandem diabody 16 redirected target cell lysiswere determined in FACS-based cytotoxicity assays with human primaryT-cells as effector cells at E:T ratios of 5:1 and 20-24 h incubation;assays with CD33-expressing CHO cells were incubated for 40-48 h. Meanand SD of at least 3 independent assays are shown.

Example 8 TandAb-Activation of T-Cells and In Vitro Killing of AML Cells

TandAbs were incubated with purified human T cells and a VPD-450-labeledhuman CD33⁺ leukemia cell line, KG-1, or the CD33⁻ human ALL cell line,G2 (E:T 5:1). Flow cytometry was used to evaluate target cell lysis byTandAbs (10⁻¹⁵ to 10⁻⁸M; 24 h, 37° C.).

Incubation of TandAbs 12, 16, and 19 with human T cells efficientlylysed KG-1 cells (IC50˜0.01, 0.5, and 5 pM respectively). Up to 40% of Tcells were activated (CD25+) rising with cytotoxic activity. A controlTandAb with an irrelevant target, 00 (>10⁻⁷ M), did not result insignificant killing of KG-1 in vitro. Separately, 16 induced lysis ofKG-1 cells (IC50=5×10⁻¹²M) while 1×10⁻⁸M had no effect on CD33− G2cells. The results indicate that T cells become activated and potentlylyse tumor cells when targeted to CD33+ leukemic cells (KG-1) andprimary CD33+ AML blasts by CD33/CD3 TandAbs.

Example 9 Epitope Mapping

Tandem diabodies containing different CD33 binding moieties weresubjected to epitope mapping using CLIPS Technology (Pepscan) in orderto identify CD33-binding epitopes.

CLIPS Technology facilitates the structuring of peptides into singleloops, double-loops, triple loops, sheet-like folds, helix-like folds,and combinations thereof, offering the possibility to map discontinuousepitopes of the target molecule.

An array of more than 7000 independent peptides was synthesized and thebinding of each antibody to the peptides was tested in an ELISA.

The tandem diabodies 12, 14, 16 and 22 bind to the stretch ₆₂DQEVQEETQ₇₀(SEQ ID NO:94) in the first Ig like domain of human CD33. The respectiveamino acid stretches are shown underlined and in bold in FIG. 9A. It iscontemplated that tandem diabodies 01, 02, 04, 06, 08, 09, 13 and 23also bind to this epitope as these tandem diabodies share the same CD33binding domains (SEQ ID NOs:2 and 12, 3 and 13, 5 and 15, 9 and 19) astandem diabodies 12, 14 16 and 12.

Example 10 Dose-Response in a Prophylactic In Vivo Tumor Model

Tandem diabodies 12 and 16 are compared at different dose levels in aprophylactic HL-60 tumor xenograft model in NOD/scid mice reconstitutedwith human T-cells. In order to achieve a dose-response three doselevels at 10, 1 and 0.1 μg (0.5, 0.05, and 0.005 mg/kg) were selected.

Eight experimental groups of immunodeficient NOD/scid mice werexenotransplanted by subcutaneous injection with a suspension of 4×10⁶HL-60 cells. Prior to injection cells were mixed with 3×10⁶ T-cellsisolated from buffy coats (healthy donors) employing negative selection.To account for potential donor variability of the T-cells, each of theexperimental groups was subdivided into three cohorts each receivingT-cells of one individual donor only. All animals of the experimentalgroups transplanted with tumor cells and T-cells received an intravenousbolus on days 0, 1, 2, 3 and 4 (qdxd5) of either vehicle (control) or 16or 12 at three different dose levels as indicated (0.1 μg, 1 μg, and 10μg). One group without effector cells and vehicle treatment served as anadditional control. Table 13 summarizes group allocation and dosingschedule.

TABLE 13 Schedule Group treatment dose Cell concentration/animal Cohort(iv) n 1 Vehicle — 4 × 106 HL-60 4 2 Vehicle — 4 × 10⁶ HL-60 + 3 × 10⁶T-cells Cohort 1 Day 0, 1, 2, 3 4 × 10⁶ HL-60 + 3 × 10⁶ T-cells Cohort 23, 4 3 4 × 10⁶ HL-60 + 3 × 10⁶ T-cells Cohort 3 3 3 16 10 μg 4 × 10⁶HL-60 + 3 × 10⁶ T-cells Cohort 1 Day 0, 1, 2, 3 4 × 10⁶ HL-60 + 3 × 10⁶T-cells Cohort 2 3, 4 3 4 × 10⁶ HL-60 + 3 × 10⁶ T-cells Cohort 3 3 4 16 1 μg 4 × 10⁶ HL-60 + 3 × 10⁶ T-cells Cohort 1 Day 0, 1, 2, 3 4 × 10⁶HL-60 + 3 × 10⁶ T-cells Cohort 2 3, 4 3 4 × 10⁶ HL-60 + 3 × 10⁶ T-cellsCohort 3 3 5 16 0.1 μg  4 × 10⁶ HL-60 + 3 × 10⁶ T-cells Cohort 1 Day 0,1, 2, 3 4 × 10⁶ HL-60 + 3 × 10⁶ T-cells Cohort 2 3, 4 3 4 × 10⁶ HL-60 +3 × 10⁶ T-cells Cohort 3 3 6 12 10 μg 4 × 10⁶ HL-60 + 3 × 10⁶ T-cellsCohort 1 Day 0, 1, 2, 3 4 × 10⁶ HL-60 + 3 × 10⁶ T-cells Cohort 2 3, 4 34 × 10⁶ HL-60 + 3 × 10⁶ T-cells Cohort 3 3 7 12  1 μg 4 × 10⁶ HL-60 + 3× 10⁶ T-cells Cohort 1 Day 0, 1, 2, 3 4 × 10⁶ HL-60 + 3 × 10⁶ T-cellsCohort 2 3, 4 3 4 × 10⁶ HL-60 + 3 × 10⁶ T-cells Cohort 3 3 8 12 0.1 μg 4 × 10⁶ HL-60 + 3 × 10⁶ T-cells Cohort 1 Day 0, 1, 2, 3 4 × 10⁶ HL-60 +3 × 10⁶ T-cells Cohort 2 3, 4 3 4 × 10⁶ HL-60 + 3 × 10⁶ T-cells Cohort 33

Treatment groups for the in vivo dose-response study in a HL-60xenograft model. All animals in the control groups reliably developed atumor and exhibited homogeneous tumor growth. The presence of T-cellshad no influence on tumor development. No difference in HL-60 growth wasobserved in the presence or absence of T-cells in the vehicle-treatedcontrol groups.

Treatment with both test items revealed a clear dose-dependentanti-tumor effect (FIG. 10). No substantial difference was found betweenthe two tandem diabodies. Plotting of mean tumor volumes in FIG. 10 wasrestricted to day 29 when most of the treatment groups were complete.The study was continued until day 45 and animals were observed fortumor-free survival. In the groups treated with 10 or 1 μg of 16, 6 of 9animals were tumor-free at the end of the observation period and 5 of 9animals receiving 10 μg of 12 were tumor-free on day 45. One animalremained tumor-free when treated with 1 μg of 12.

All animals in the control groups reliably developed a tumor andexhibited homogeneous tumor growth. Treatment with either of the tandemdiabodies revealed a dose-dependent anti-tumor effect and no substantialdifference was found between the two tandem diabodies until day 29.

Detectable differences were observed only after prolonged observation(day 45), at which time the low dose and control groups had already beenterminated due to the growth of large tumors. Groups treated with 16 hadmore tumor-free animals.

Example 11 Established Tumor Model

A xenograft model in NOD/scid mice with pre-established HL-60 tumorsemploying 16 was developed to demonstrate proof of concept.

In brief, female immune-deficient NOD/scid mice were sub-lethallyirradiated (2 Gy) and subcutaneously inoculated with 4×10⁶ HL-60 cells.On day 9 the animals received a single bolus injection of anti-asialoGM1 rabbit antibody (Wako, Neuss, Germany) to deplete murine naturalkiller (NK) cells. On day 10, when the tumor reached a volume between50-150 mm³ (mean 73±11 mm³) animals were allocated to 3 treatmentgroups. Groups 2 and 3 (8 animals each) were intraperitoneally injectedwith 1.5×10⁷ activated human T-cells. Prior to injection T-cells wereisolated from buffy coats (healthy donors) employing negative selection.T-cells were expanded and activated with the T-Cell Activation/ExpansionKit according to the manufacturer's specification (Miltenyi Biotech). Inorder to address potential donor variability Groups 2 and 3 weresubdivided into two cohorts each receiving expanded and activatedT-cells from an individual donor. Each cohort received T-cells from oneindividual T-cell donor only.

TABLE 14 Treatment groups for the established HL-60 xenograft model.Animals Inoculated cells Treatment Group (n) Day 0, sc. Day 10, ip.Cohort Day 13 to 21, once daily 1 5 4 × 10⁶ HL-60 Vehicle (iv) 2 4 4 ×10⁶ HL-60 1.5 × 10⁷ T-cells (Donor 1) 1 Vehicle (iv) 4 4 × 10⁶ HL-60 1.5× 10⁷ T-cells (Donor 2) 2 3 4 4 × 10⁶ HL-60 1.5 × 10⁷ T-cells (Donor 1)1 TandAb 16 (iv) 50 μg 4 4 × 10⁶ HL-60 1.5 × 107 T-cells (Donor 2) 2

Starting on day 13 animals in Group 3 displayed a mean tumor volume of105 mm³ and were treated with a total of 9 intravenous doses of 50 μgtandem diabody 16 (qdx9d). Table 14 illustrates group allocation anddosing schedule. Groups 1 and 2 were only treated with the vehicle. Bodyweight and tumor volume were determined until day 27.

All animals reliably developed a tumor, which was palpable on day 6. Themean tumor volume of vehicle-treated Group 1 and 2 (HL-60) animalscontinually increased until study termination on day 27 (FIG. 11). InGroup 2 animals that received primary activated human T-cells inaddition to HL-60 tumor cells, the mean tumor volume increased fastercompared to Group 1 (HL-60 only).

Repeated intravenous treatment from days 13 to 21 (qdxd9) with tandemdiabody 16 (50 μg/animal; 2.5 mg/kg) in the presence of human T-cells(Group 3) rapidly delayed tumor growth relative to Group 1 and Group 2.Tandem diabody 16 delayed tumor growth in Group 3 by approximately 4-5days compared to vehicle-treated control group (Group 2). Statisticallysignificant differences in the time period from day 6 to day 27 wereidentified between Group 2 (HL-60, T-cells, vehicle) and Group 3 (HL-60,T-cells, 16) on day 22 (p<0.05), day 23 (p<0.01) and day 27 (p<0.01)(Two-way Repeated Measures ANOVA with Bonferroni post-tests). Nostatistically significant differences were present between Group 1 andGroup 3 due to unusual slow growth of the tumor in Group 1.

No donor variability with regard to T-cell activity was observed, whencomparing tumor development in Cohort 1 and Cohort 2 within a group,which received T-cells from different donors (see Table 14).

Example 10 shows that a xenograft model in NOD/scid mice with apre-established HL-60 tumor (AML) and intraperitoneally-engrafted humanT-cells was successfully developed. Repeated dosing with tandem diabody16 at a single dose level lead to a statistically significant delay intumor growth in comparison to the respective vehicle-treated controlgroup. The data generated are comparable to results published for asimilar study with a CD33/CD3 BiTE™ (Aigner et al., 2012; Leukemia,2013, April; 27(5):1107-15).

Example 12 Efficacy of CD33/CD3 Tandem Diabodies in an AML PDX Model inNSG Mice

Cryopreserved cells from an AML patient whose CD33⁺ leukemia contained2-4% CD3⁺ T-cells were used to establish an AML PDX model in NSG mice.One hour post-injection of tumor cells into irradiated (250 cGy) NSGmice, CD33/CD3 tandem diabodies, 16 or 12, at either of two i.v. doses(50 μg or 5 μg; n=8 mice/group) were injected in a 200 μL bolus.Additional injections of tandem diabodies were performed on each of thefollowing 4 days. Mice were weighed once weekly, and subsequently weresacrificed on day 38 to permit collection of peripheral blood, bonemarrow, and spleen for analysis by flow cytometry (huCD33, huCD34,huCD45, muCD45, huCD14, huCD3, huCD4, huCD8, and 7AAD). The results areshown in FIG. 12.

FIG. 12 shows that untreated mice had substantial amounts of humanblasts in the bone marrow and spleen after 38 days. In contrast, micetreated with daily i.v. injections of tandem diabodies 12 or 16exhibited substantially lower numbers of human AML blasts in the bonemarrow and in the spleen. The strong anti-AML effect of the CD33/CD3tandem diabody was observed at both dose levels (5 and 50 μg/injection).

The observed anti-AML effect for both CD33/CD3 tandem diabodies, 12 and16, was much stronger than the effect of a CD123/CD3 DART® antibodytargeting AML in an identical mouse model (Hussaini et al.: “TargetingCD123 In Leukemic Stem Cells Using Dual Affinity Re-Targeting Molecules(DARTs®) Nov. 15, 2013; Blood: 122 (21)). In contrast to the CD33/CD3tandem diabodies which eliminated nearly all AML blasts in bone marrowand spleen, Hussaini et al. reported that the CD123/CD3 DART® reducedthe number of AML blasts in the bone marrow and spleen in the PDX modelonly by factor 50-1000 at 2.5 and 0.25 mg/kg, the authors furtherreported that the CD123/CD3 DART™ reduced the number of AML blasts inbone marrow and spleen in the PDX model only by 40-78% at 0.5 mg/kg.

Example 13 Fast Onset of CD33/CD3 Tandem Diabody 16-Mediated Target CellLysis

In order to assess the kinetics of CD33/CD3 tandem diabody-mediatedtarget cell lysis, calcein-release cytotoxicity assays with differentincubation times were performed. Calcein-labeled CD33⁺ HL-60 targetcells were incubated with serial dilutions of tandem diabody 16 in thepresence of primary human T cells as effector cells at an E:T ratio of25:1 for 30 min, 1 h, 2 h, 3 h, 4 h, or 5 h. At each time point thecalcein that was released from lysed target cells was used to calculatethe EC₅₀ value and tandem diabody 16-mediated target cell lysis usingnon-linear regression/sigmoidal dose-response. FIG. 13 shows anunexpected fast onset of tandem diabody-mediated target cells lysis withmore than 40% lysis after 30 min incubation at saturating tandem diabodyconcentrations. After 4 hours incubation more than 90% target cell lysiswas reached. Table 15 and FIG. 14 summarize the EC₅₀ and specific lysisvalues determined for tandem diabody 16 at incubation times between 30min and 5 hours. The results further demonstrate that under the usedassay conditions maximal potency (lowest EC₅₀ value) was reached after 2hours incubation and that after 5 hours incubation almost all targetcells were lysed. Altogether these results demonstrate a very fast,potent and efficacious target cell lysis mediated by CD33/CD3 tandemdiabodies.

TABLE 15 Kinetics of EC₅₀ and lysis values determined for tandem diabody16 incubation tandem diabody- time [min] EC₅₀ [pM] mediated lysis [%] 304.8 44.1 60 2.5 59.8 120 1.6 75.1 180 1.6 88.8 240 1.5 93.7 300 1.6 97.4

Example 14 Proof-of-Concept Clinical Trial Protocol for Administrationof CD33/CD3 Tandem Diabodies to AML Patients

This Phase I/II clinical trial for studying CD33/CD3 tandem diabody 16as a treatment for with acute myeloid leukemia (AML).

Study Outcomes:

Primary:

Maximum tolerated dose of CD33/CD3 tandem diabody 16

Secondary:

To determine whether in vitro response of CD33/CD3 tandem diabody 16 isassociated with clinical response

Phase I

The maximum tolerated dose (MTD) will be determined in the phase Isection of the trial.

-   -   1.1 The maximum tolerated dose (MTD) will be determined in the        phase I section of the trial.    -   1.2 Patients who fulfill eligibility criteria will be entered        into the trial to CD33/CD3 tandem diabody 16.    -   1.3 The goal is to identify the highest dose of CD33/CD3 tandem        diabody 16 that can be administered safely without severe or        unmanageable side effects in participants. The dose given will        depend on the number of participants who have been enrolled in        the study prior and how well the dose was tolerated. Not all        participants will receive the same dose.

Phase II

-   -   2.1 A subsequent phase II section will be treated at the MTD        with a goal of determining if therapy with therapy of CD33/CD3        tandem diabody 16 results in at least a 20% response rate.    -   Primary Outcome for the Phase II—To determine if therapy of        CD33/CD3 tandem diabody 16 results in at least 20% of patients        achieving a clinical response (blast response, minor response,        partial response, or complete response)

Eligibility:

-   -   Documented AML by peripheral blood and bone marrow analyses        meeting WHO criteria, excluding patients with acute        promyelocytic leukemia (APL)        -   Patients with AML refractory to primary induction            chemotherapy, relapsed disease, or age ≥60 and not            appropriate for standard cytotoxic therapy due to age,            performance status, and/or adverse risk factors according to            the treating physician        -   Age ≥18 years        -   Karnofsky performance status ≥50% or ECOG performance status            0-2        -   Life expectancy ≥6 weeks

While certain embodiments have been shown and described herein, it willbe obvious to those skilled in the art that such embodiments areprovided by way of example only. Numerous variations, changes, andsubstitutions will now occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments described herein may be employed inpracticing the embodiments. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

1.-30. (canceled)
 31. A CD33 binding protein specific to human CD33 and human CD3, wherein said CD33 binding protein comprises a tandem diabody comprises a variable light chain (VL) domain and a variable heavy chain (VH) domain and wherein, the VL domain comprises a CDR1 consisting of the sequence selected from the group consisting of SEQ ID NOs:21-27, a CDR2 consisting of the sequence selected from the group consisting of SEQ ID NOs:28-34 and a CDR3 consisting of the sequence of the group consisting of SEQ ID NOs:35-41, and the VH domain comprises a CDR1 consisting of the sequence selected from the group consisting of SEQ ID NOs:42-48, a CDR2 consisting of the sequence selected from the group consisting of SEQ ID NOs:49-55 and a CDR3 consisting of a sequences selected from the group consisting of SEQ ID NOs:56-63.
 32. The CD33 binding protein according to claim 31, wherein the CDR1, CDR2 and CDR3 of the VL domain are sequences selected from the group consisting of: (i) SEQ ID NOs:21, 28 and 35; (ii) SEQ ID NOs:22, 29 and 36; (iii) SEQ ID NOs:23, 30 and 37; (iv) SEQ ID NOs:24, 31 and 38; (v) SEQ ID NOs:25, 32 and 39; (vi) SEQ ID NOs:26, 33 and 40; and (vii) SEQ ID NOs:27, 34 and
 41. 33. The CD33 binding protein according to claim 31, wherein the CDR1, CDR2 and CDR3 of the VH domain are sequences selected from the group consisting of: (i) SEQ ID NOs:42, 49 and 56; (ii) SEQ ID NOs:43, 50 and 57; (iii) SEQ ID NOs:43, 50 and 58; (iv) SEQ ID NOs:43, 50 and 59; (v) SEQ ID NOs:43, 50 and 60; (vi) SEQ ID NOs:44, 51 and 61; (vii) SEQ ID NOs:45, 52 and 62; (viii) SEQ ID NOs:46, 53 and 63; (ix) SEQ ID NOs:47, 54 and 63; and (x) SEQ ID NOs:48, 55 and
 63. 34. The CD33 binding protein according to claim 31, wherein the VL and VH domains are sequences selected from the group consisting of: (i) SEQ ID NO:1 and SEQ ID NO: 11; (ii) SEQ ID NO:3 and SEQ ID NO: 13; (iii) SEQ ID NO:4 and SEQ ID NO: 14; (iv) SEQ ID NO:5 and SEQ ID NO: 15; (v) SEQ ID NO:6 and SEQ ID NO: 16; (vi) SEQ ID NO:7 and SEQ ID NO: 17; (vii) SEQ ID NO:8 and SEQ ID NO: 18; (viii) SEQ ID NO:9 and SEQ ID NO: 19; and (ix) SEQ ID NO: 10 and SEQ ID NO:20.
 35. The CD33 binding protein according to claim 31, wherein the CD33 binding protein binds to amino acid residues 62-70 of human CD33 (SEQ ID NO:93).
 36. The CD33 binding protein according to claim 31, wherein the CD33 binding protein further comprises an effector domain.
 37. The CD33 binding protein according to claim 36, wherein the effector domain comprises a binding site specific for an effector cell.
 38. The CD33 binding protein according to claim 37, wherein the effector cell is a T-cell.
 39. The CD33 binding protein according to claim 37, wherein the binding site comprises a VL and VH domain that binds to human CD3.
 40. The CD33 binding protein according to claim 39, wherein the VH domain specific for human CD3 comprises a CDR1 sequence of STYAMN (SEQ ID NO:72), a CDR2 sequence of RIRSKYNNYATYYADSVKD (SEQ ID NO:73) and a CDR3 sequence of HGNFGNSYVSWFAY (SEQ ID NO:74) or HGNFGNSYVSYFAY (SEQ ID NO:75).
 41. The CD33 binding protein according to claim 39, wherein the VL domain specific for human CD3 comprises a CDR1 sequence ofRSSTGAVTTSNYAN (SEQ ID NO:90), a CDR2 sequence of GTNKRAP (SEQ ID NO:91), and a CDR3 sequence of ALWYSNL (SEQ ID NO:92).
 42. The CD33 binding protein according to claim 39, wherein the VL and VH domains specific to CD3 are sequences selected from the group consisting of: (i) SEQ ID NO:64 and SEQ ID NO:68; (ii) SEQ ID NO:65 and SEQ ID NO:69; (iii) SEQ ID NO:66 and SEQ ID NO:70; and (iv) SEQ ID NO:67 and SEQ ID NO:71.
 43. The CD33 binding protein according to claim 37, wherein the effector domain is linked by a linker that consists of about 12 or less amino acid residues.
 44. The CD33 binding protein according to claim 43, wherein the is selected from GGSGGS (SEQ ID NO:95), GGSG (SEQ ID NO:96) or GGSGG (SEQ ID NO:97). 